NURSERY MAINTENANCE OF THE PRAWN, MACROBRACHIUM ROSENBERGII

EARLY POST-LARVAE, ASSESSMENT OF THE FEASIBILITY OF INDIAN

BORAGE LEAF, PLECTRANTHUS AMBOINICUS AS A FEED INGREDIENT FOR
GROWTH OF M. ROSENBERGII POST-LARVAE, AND EVALUATION OF THE
ANTIOXIDANT POTENCY OF INDIAN BORAGE AND ITS REACTIVE OXYGEN
SPECIES REDUCTION CAPACITY IN ARTEMIA NAUPLII

Dissertation submitted to the Bharathiar University in partial fulfillment of the

requirements for the award of the Degree of
MASTER OF SCIENCE IN ZOOLOGY

Submitted by
ARUNKUMAR G
(Reg. No. 20ZOOD05)

Under the Guidance of

Dr. P. SARAVANA BHAVAN
Professor

CRUSTACEAN BIOLOGY LABORATORY

DEPARTMENT OF ZOOLOGY
SCHOOL OF LIFE SCIENCES
BHARATHIAR UNIVERSITY
COIMBATORE – 641046

MAY 2022
CERTIFICATE

This is to certify that the dissertation entitled, “NURSERY MAINTENANCE OF THE

PRAWN, MACROBRACHIUM ROSENBERGII EARLY POST-LARVAE,
ASSESSMENT OF THE FEASIBILITY OF INDIAN BORAGE LEAF,
PLECTRANTHUS AMBOINICUS AS A FEED INGREDIENT FOR GROWTH OF M.
ROSENBERGII POST-LARVAE, AND EVALUATION OF THE ANTIOXIDANT
POTENCY OF INDIAN BORAGE AND ITS REACTIVE OXYGEN SPECIES
REDUCTION CAPACITY IN ARTEMIA NAUPLII”, submitted to Bharathiar University,
Coimbatore in partial fulfillment of the requirements for the award of the Degree of MASTER
OF SCIENCE IN ZOOLOGY is a record of original research work done by ARUNKUMAR
G during the period (2021-2022) of her research in the Department of Zoology, Bharathiar
University, Coimbatore, under my supervision and guidance and dissertation has not formed
the basis for the award of any Degree / Diploma / Associateship / Fellowship or other similar
title of any candidate of any other University.

Date:

Place: Coimbatore

Countersigned Signature of the Guide

Head of the Department

DECLARATION

I, ARUNKUMAR G, hereby declare that the dissertation entitled, “NURSERY

MAINTENANCE OF THE PRAWN, MACROBRACHIUM ROSENBERGII
EARLY POST-LARVAE, ASSESSMENT OF THE FEASIBILITY OF INDIAN
BORAGE PLECTRANTHUS AMBOINICUS, AS A FEED INGREDIENT FOR
GROWTH OF M. ROSENBERGII POST-LARVAE, AND EVALUATION OF THE
ANTIOXIDANT POTENCY OF INDIAN BORAGE AND ITS REACTIVE
OXYGEN SPECIES REDUCTION CAPACITY IN ARTEMIA NAUPLII”, submitted
to Bharathiar University, Coimbatore in partial fulfillment of the requirements for the award
of Degree of MASTER OF SCIENCE IN ZOOLOGY is a record of original research work
done by me during 2021- 2022 under the supervision and guidance of Dr. P. SARAVANA
BHAVAN, Professor, Department of Zoology, Bharathiar University, Coimbatore, and it
has not formed the basis for the award of any Degree / Diploma / Associateship /
Fellowship or other similar title to any candidate of any other University.

Date: Signature of the Candidate

Place: Coimbatore (ARUNKUMAR G)
ACKNOWLEDGEMENT

My heart beating thanks to my beloved mentor Dr. P. Saravana Bhavan, professor,

Department of Zoology, Bharathiar University, Coimbatore who made this happen by adding
his valuable insights into my dissertation but also handled every circumstance with his valuable
possession called intellectual humility

With a deep sense of gratitude, I express my personal indebtedness and many thanks to
all the teaching faculty of Zoology department, Dr. M. Ramesh, Professor and Head, Dr. V.
Ramasubramaniam, Professor, Dr. C. Gunasekaran, Associate Professor Dr. N. Arul,
Assistant Professor, Dr. T. Muralisankar, Assistant Professor, Dr.Vivek, Ramalinga
Swamy re-entry fellow, Bharathiar University for their suggestions and guidance during
the period of my study.

I would also like to thank the administrative staff members of Department of

Zoology, Dr. V. Maruthappan, ATO, Mr. Senthil Kumaresan, Mrs. Amutha, ROA, and
J. Karthick Balaji for the technical assistance in time during the course of study and
aiding me at right times.

My profound thanks to our lab seniors, Ms. R. kalpana, Ms. T. Manjula, Ms. C.
Dharani, Mr. S. Ramachandra prabhu, Ms. M. Harini, Mr. G. Thiruppathi, for their
hearty valuable guidance and most help rendered during my study.

I would like to thanks my lab friends Ms.kaviya.V, Ms.Santhiya.S,

Ms.Pavithra devi. K, Ms.Durga.U their encouragement helped me to reach the shores
of success.

I would like to give my lovable thank to my parents, and my friends for their
encouragement and moral support which made me to shine to every minute of my life
time.

(G.ARUNKUMAR)
S. No TITLE PAGE
No

1 Introduction

1.1. Aquaculture

1.2. Crustaceans in Aquaculture

1.3. F Freshwater Prawn Culture

1.4 Macrobrachium rosenbergii

1.5 Life cycle of Macrobrachium rosenbergii

1.6 Aquaculture Nutrition

1.7 Natural and Live feeds

1.7.1 Biology of brine shrimp, Artemia franciscana

1.8 Indian borage (Plectranthus amboinicus)

1.8.1 Taxonomy of Plectranthus amboinicus

1.8.2 Morphological features of P.amboinicus

1.8.3 Origin and Geographical distribution of P.amboinicus

1.8.4 Nutritional value of P.amboinicus

1.8.5 Uses of P.amboinicus

1.9 Aim and Objectives

2 Materials and Methods

2.1 Procurement of acclimatization of M.rosenbergii early post

larvae PL
2.1.1 Determination of growth performance of M.rosenbergii
early PL

2.2 Collection and Processing of Indian borage leaves

2.3 Analysis of Proximate composition of Indian borage leaf powder

2.4 Preparation of extracts (Soxhlet and Rotary evaporation)

2.5 Analysis of the principle secondary phytochemicals of the

methanolic extract P.amboinicus leaves

2.6 Feed preparation

2.6.1 Feeding trail

2.6.2 Survival, Growth, Nutritional indices

2.7 In vitro antioxidant of Indian borage leaf powder

2.7.1 DPPH radical scavenging assay

2.8 ROS reduction capacity of Indian borage leaf powder in Artemia

nauplii

2.8.1 Enrichment

2.8.2 Intracellular ROS generation in Artemia nauplii

3 Results

3.1 Nursery maintenance of M.rosenbergii early PL

3.2 Proximate composition of Indian borage leaf powder

3.3 The principle secondary phytochemicals of the methanolic extract

of Indian borage leaf powder

3.4 Survival, Growth, and Nutritional Indices

3.5 DPPH free radical scavenging activity of P. amboinicus leaf
powder

3.6 Enrichment of Artemia nauplii with P. amboinicus leaf powder

3.7 ROS reduction capacity of Indian borage leaf powder in Artemia

nauplii

4 Discussion

4.1 Growth and Survival rate ( In nursery maintenance)

4.2 Proximate composition of Indian borage leaf powder

4.3 Formulated diets

4.4 Growth and Survival rate (In experimental PL)

4.5 DPPH radical scavenging activity

4.6 ROS (Reactive Oxygen Species)

5 Summary and Conclusion

6 References

7 7.1 Annexure – I

7.2 Annexure – II
NURSERY MAINTENANCE OF THE PRAWN, MACROBRACHIUM
ROSENBERGII EARLY POST-LARVAE, ASSESSMENT OF THE FEASIBILITY
OF INDIAN BORAGE LEAF, PLECTRANTHUS AMBOINICUS AS A FEED
INGREDIENT FOR GROWTH OF M. ROSENBERGII POST-LARVAE, AND
EVALUATION OF THE ANTIOXIDANT POTENCY OF INDIAN BORAGE AND
ITS REACTIVE OXYGEN SPECIES REDUCTION CAPACITY IN ARTEMIA
NAUPLII

1. INTRODUCTION

1.1. Aquaculture

Aquaculture is the regulated production of freshwater or saltwater creatures, and it is one

of the world's most developed activities. Aquaculture currently provides half of the fish and
mollusks required to feed the world's population (FAO 2018). It provides an essential
resource for meeting the rising food demand and addressing nutritional deficiencies (Golden
et al., 2021; Smith et al., 2010; Filipski and Belton, 2018), It is the fastest growing food
production system (Garlock et al., 2020). Fish and other aquatic foods are nutrient dense
animal food sources; they are from marine and freshwater environments. Bio available
minerals such as zinc, calcium, and long-chain n-3 polyunsaturated fatty acids are present in
aquatic diet (Rimm et al., 2018; Hibbeln et al., 2019; Mohan et al., 2021). Aquaculture is
a well-founded, animal food producing sector in the world. Globally, the average annual per
capita consumption is 20.21kg. In some Asian countries, the rate of culture of aquatic
organisms has been increased, For example, since 1990; Chinese consumption of aquatic
food has more than tripled, rising from 11.2 kg per capita per year to 38.0 kg per capita per
year in 2018. It is an effort for the sustainable development of aquaculture in order to fulfill
future demands from a world population of 9.6 billion people by 2050.

India is the world's second-largest fish producer, with 7.58 percent of global output.
During 2019-20, India's fish production reached an all-time high of 14.16 million metric
tonnes. Indian aquaculture production can be classified into freshwater and brackish water.
The support for the development and growth of freshwater and coastal aquaculture is
provided by the Indian Government through a network of 429 Fish Farmers Development
Agencies (FFDA) and 39 Brackish Fish Farmers Development Agencies (BFDAs)
(Ayyappan, 2014). During 2019-20, marine products export totaled 12.9 lakh metric tones,
with a value of Rs 46,662 crores. Over 28 million people's incomes have been sustained.
Thanks to the livelihood provided by this sector, in India, particularly among the poor and
vulnerable, for promoting substantial change.

1.2. Crustaceans in Aquaculture

Crustaceans are biologically diverse, ancient, globally distributed, and experimentally

tractable (VanHook and Patel 2008). Crustacea represent one of the largest extant taxon in
the animal kingdom, and this taxon has evolved an impressing variety of life styles, body
shapes, colors, and sizes. It comprises pelagic oceanic prawns, freshwater crabs and
crayfish, tiny pea crabs living hidden inside the mantle (Provenzano 1985). Crustacean
represents a large group of segmented body animals with exoskeleton and characteristic
double pair of antennae, although also including a few freshwater and terrestrial species.
This group has an extensive fossil record, reaching back to the Cambrian. In adult
crustaceans, the exoskeletal cuticle is organized in three principle layer: epicuticle ,
exocuticle, and endocuticle (Compare et al., 2004; Dillaman et al., 2013).

The crustacean aquaculture rapidly emerging world industry, amidst increasing concerns
about animal protein sources, employment and income sources, and foreign exchange gain,
for example, in 2013, of 19.7 kg of fish per capita available for consumption; approximately
1.8 kg was obtained from crustaceans (FAQ, 2016). Global crustacean production was 5.4
million tons in 2010, and reached 8.4 million tons in 2017.

In India, Crustaceans are one of the most valuable resources in the marine fishery and
contributed an overall average of 14.9 % to the total landings during 1996-2019. Penaeid
and non-penaeid prawns, crabs, lobsters, and stomatopods are the most common marine
commercial crustaceans.

1.3. Freshwater Prawn Culture

Freshwater prawns have been reared in captivity, either through introducing wild-caught
juveniles or by trapping them, along with other crustaceans. The culture of Macrobrachium
rosenbergii has an vital role in freshwater prawn culture. The giant freshwater prawn, M.
rosenbergii is a common inhibitant of river and indigenous to the Indo-Pacific region
(Aflalo et al., 2012; Gao et al., 2020; Jiang et al., 2020; Mostafiz et al., 2020).This
species with high protein content, tasty flavor, and larger size, M. rosenbergii has become a
global food source (Banu and Christianus, 2016; Rabiul Islam et al., 2017; Roustaian et
al., 1999; Roustaian et al., 2000). The optimum temperature and optimum pH range for the
growth of M. rosenbergii are 29-31̊ C and 7.0-8.5, respectively. Prawn which are benthic,
omnivorous, produced in ponds, they consumes feed as commercial feed, bottom
invertebrates, detritus, wastes and feces (Santos and Valenti, 2002; D́ Abramoand New,
2010). In fresh water pond, pH level fluctuate from 6.6 to 10.2 because of the removal of
carbon dioxide due to photosynthesis of plants during daytime and the release of CO2 by
both plants and animals during the night.
India is the second-largest contributor of freshwater prawns to the world market. West
Bengal is the biggest producer, followed by Orissa and Andhra Pradesh, with a total prawn
production of 3332 metric tonnes from 2919 ha and an average productivity of 1.14 metric
tonnes / hectare / year (Bavithra et al., 2012). Growing prawn in ponds is becoming more
popular as it is a more practical approach than catching them in lakes, rivers, canals,
streams, or estuaries. In freshwater ponds, prawns grow quickly, reaching marketable size
(150-180 mm) in about six months. In fertilized pond they grow very fast. It can be built
wherever the soil, shape of the land and water supply are appropriate. Through the pond
prawn culture leads to high production and it has many problems, such as polluted
wastewater, poor product, muscle quality and susceptibility to disease, are related with it.
These problems results in economic losses for farmers and the environment (Heldt et al.,
2019; Wu et al., 2017;Huang et al., 2014). In neighboring nations like Bangladesh, Sri
Lanka, and Pakistan, prawn culture has a lot of potential.

1.4. Classification and Biology of Macrobrachium rosenbergii

Phylum : Arthropoda
Subphylum : Crustacea
Class : Malacostraca
Subclass : Eumalacostraca
Superorder : Eucarida
Order : Decapoda
Sub-order : Natantia
Super family : Palaeomonoidea
Family : Palaemonidea
Genus : Macrobrachium
Species : rosenbergii (De Man, 1879)
The giant freshwater prawn, Macrobarchium rosenbergii (De Man, 1879), is the world’s
largest palaemonid prawn and it was called as scampi. M. rosenbergii was indigenous to the
Indo-Pacific region and found mostly in Malaysia, Thailand, India, Bangladesh, and
Myanmar (Aflalo et al., 2012; Gao et al., 2020; Jiang et al., 2020; Mostafiz et al., 2020).
M. rosenbergii was initially introduced to China in 1976, and as a result of its wonderful
taste and great economic worth, production has continued to grow and develop (Yang et al.,
2012). M. rosenbergii cultivated output in China was 139,609 metric tonnes in 2019,
accounting for 1,212,783,000 USD, making it the world’s largest M. rosenbergii production
(FAO, 2021). Furthermore, the production volumes for Indonesia, Malaysia, Myanmar,
Philippines, Thailand, and Bangladesh were 4,600, 205.58, 9,509.07, 1.24, 31,345, and
52,197 metric tonnes, respectively (FAO, 2021). With rising consumer demand and
increased production, M. rosenbergii aquaculture has significant hurdles in terms of
broodstock management, embryonic development, and larviculture.
There are a number of biological and ecological characteristics that might explain its
potential invasive nature. First of all, M. rosenbergii is characterized by a very high
fecundity, with c. 80 000 eggs per female (the highest in the genus), compared to closely
related species such as M. amazonicum with c. 2000 eggs per female (Da Silva et al., 2004).
Most of the species spend their early life in brackish water that is connected directly with
sea. Some species complete their lifecycle in freshwater but these are not of commercial
importance. Macrobrachium rosenbergii is most suitable for culture due to its less
aggressive nature under culture conditions. There are 150 species of Macrobrachium in
world, of which 49 are commercial. Twenty seven of the commercial species are found in
Asia and the Pacific. Most live in freshwater, 25 species are found in India
(Soundarapandian and Kannan, 2008).

1.5. Life cycle of M.rosenbergii

There are four distinct phases in the life cycle of M.rosenbergii, namely, eggs, larvae,
postlarvae and adults. The life cycle of M.rosenbergii summarized as follows.
Breeding
Females generally become reproductively mature within 6 months of age. Mating can
only occurs between hard-shelled male and soft shelled females. Within few hours to molt,
eggs are released and fertilized by sperms. The fertilized eggs are transferred to the
underside of the abdominal in a brood chamber, formed by swimming appendages.
Egg
The eggs of M.rosenbergii are slightly elliptical, long axis measures 0.6-0.7 mm, bright
orange in colour, until 2-3 days before hatching when they become grey-black. The female
M. rosenbergii has been reported to lay up to 1,00,000 eggs in one spawning when fully
matured. The bright yellow color of the newly spawned eggs gradually changes to orange,
then brown and finally to grey about 2 to 3 days before hatching. Newly hatched nauplius
the enter into a larval phase.
Larvae
After hatching larvae are released swim upside down. They are aggressive sight feeders
and feed on small zooplankton, large phytoplankton and larval stages of aquatic
invertebrates. Larvae undergoes 11 moults, following the last moult, the larvae transform
into the post larvae. The time duration depends upon the food quality and quantity,
temperature, light and variety of water quality.
Post larvae
After metamorphosis to post larvae having total body length of 7-10 mm and weighting
6-9 mg. When they also swim, move like adults, with the dorsal side upper most and in a
head forward direction. It migrate to freshwater. Post larva are translucent and as they
change to juvenile stages. They took on the bluish green to brownish color of the adult
stages.
Adult
The adult male and female was easily distinguishable. Older juvenile and adult usually
have a distinctive blue green color, although sometimes they may taken on a brownish blue,
color is usually the result of the type of diet. The matured male and female introduced to
mating. The flops or clear "bubbles" that cover the gonophores through which sperm are
discharged are formed when the base of a male's fifth or last pair of walking legs (periods) is
extended inward. Females have a genital hole at the base of each of the third pair of walking
legs, whilst males have a broad gap between the last pair of walking legs.
Based on outward traits, three distinct categories of adult males have been discovered.
Blue claws (BC) males are easily identifiable due to their lengthy, spiky blue claws. There
are two other non-blue claws morph kinds. Males with orange claws (OC) or strong orange
claws (SOC). OC and SOC men are produced by the transformational sequence. The entire
body weight of certain smaller OC males grows relatively slowly. However, they play a
larger role in reproductive activity than other OC males.
BC males and some of the smaller OC males are the most reproductively active and most
successful at mating. The BC male maintains a territory associated with a group of females
that are ready for mating and protect them during a vulnerable period, just before and after
molting. Small OC males will eventually grow and advance to the SOC stage and finally
into BC, former males undergo an extended period of non-molting (an-ecdysis), as the BC’s
reproductive capacity diminishes. Eventually, the BC males molt and return to a growth
phase during which its reproduction capacity is renewed (Louis et al., 1998

1.6. Aquaculture Nutrition

Nutrition and feeding are vital to the long sustainability of crustacean aquaculture
(Harrison, 1997). Feed is a major concern for shrimp farmers, representing up to 60% of
the total variable production costs (Akiyama et al., 1992). For several fish and shrimp
species, dietary protein, lipid requirements, and carbohydrate utilization have been well
studied. However, data on micronutrient requirements such as amino acids, fatty acids, and
minerals are only available for the most commonly cultivated carnivorous and selected
omnivorous fish species.

1.7. Natural and Live Feeds

Natural live feed organisms are a vital resource in aquaculture operations. They are a
better alternative than artificial feed during larval stages because of their ability to migrate
along all water columns, smaller size, repeatability, and higher nutritional levels. These
organisms are enriched with most essential micro and macro nutrients, viz., essential
proteins, lipids, carbohydrates, vitamins, minerals, amino acids and fatty acids (New,
1998), thus are nutritionally balanced. Despite the large scale usage of artificial feed
throughout the world, natural live feed has been found to be essential for proper growth
of juvenile forms owing to their higher nutritional values and acceptance (Gogoi et al.,
2016).

In the tropical countries, including India, natural live feeds mainly comprise of
two components: algal and non-algal. Non-algal components comprise of brine shrimps
(Artemia sp.), rotifers, freshwater cladocerans, copepods and their larvae
(Palanichamy,1996;Gogoi et al.,2016; Radhakrishnan Kandathil et al., 2020). Choosing
appropriate live feed organism at optimum life cycle stage of fish larvae requires the
consideration of the following criteria: size of the feed, gape-size of the fry or fingerling’s
mouth; the nutritional quality of the of the feed and nutritional requirement of the larvae; the
feed should essentially be rich in highly unsaturated fatty acids (HUFA); Feed organism
should reproduce fast and increase in number; and also should be sturdy and eurytolerant
(Anuraj et al., 2015). The two major live feed organisms Artemia sp.and Cladocera is
pertinent to highlight their suitability in commercial aquaculture.

1.7.1. Biology of the brine shrimp, Artemia franciscana

Scientific classification

Phylum : Arthropoda

Class : Branchiopoda

Order : Anostraca

Family : Artemiidae

Genus : Artemia

Species : franciscana (Kellogg, 1906)

Artemia franciscana, first found at Redwood City, San Francisco Bay, has since been
identified as an invasive species in numerous countries (Kellogg, 1906; Sheir et al., 2018).
Artemia is a critical species as prey for larval fish in aquaculture and an experimental
organism in ecotoxicology (Hwang et al., 2010; Lenor-mand et al., 2018). A. franciscana
cysts are estimated to account for up to 90% of the global Artemia trade (Treece, 2000).
Artemia is bisexual or parthenogenetic filter feeders that eat phytoplanktons and prokaryotes
in high-salinity environments. Artemia species must be severe osmoregulators because they
often consume high-salinity water alongside their prey (Copeland, 1967; Holliday et al.,
1990; Bradley, 2009; Sellami et al., 2020). In nature, Artemia are normally found in hyper
saline lakes ad lagoons and man-made salterns. Brine Shrimp thrive very well in natural
seawater and can tolerate salinity ranges from 3 to 300 parts per thousand. Different
geographical strains have adapted to widely fluctuating conditions with regard to
temperature (6 – 35oC), salinity and ionic composition of the biotype (Van Stappen, 2007).

The global distribution of brine shrimp in a variety of isolated habitats, each with its own
ecological conditions, has resulted in the existence of numerous geographical strains, or
genetically different populations within the same sibling species; in particular, the Artemia
parthenogenetica with its tri-, tetra-, and pentaploid populations exhibit a wide genetic
variability as well as a unique diversity in various quantitative characterists. Some of these
traits (for example, the nutritional value of newly hatched nauplii) are phenotypical,
meaning they vary from year to year or season to season (Amin & Wink, 2014). Artemia
biotopes have a relatively simple tropical structure and low species diversity; the lack of
predators and food competition allows brine shrimp monocultures to form. The Artemia first
larval stage (instar I: 400-500 m in length) is brownish-orange in color, with three pairs of
appendages and a red nauplius eye in the head area. The animal molts into the second larval
stage after around 8 hours (instar II). The 2nd antennae filter out small food particles (e.g.
algae cells, bacteria, detritus) ranging in size from 1 to 50 m and consume them into the
functional digestive system.

1.8. Indian borage (Plectranthus amboinicus)

Medicinal plants represent a vast reservoir of natural bioactive compounds widely

appreciated and utilized in traditional medicines. . A certain number of modern medicinal
drugs are produced using natural products, for instance, medicinal plant-derived
chemotherapeutic analogs such as vinblastine and topotecan. Plectranthus amboinicus is an
interesting and potentially rewarding therapeutic herb. P. amboinicus, often known as Indian
borage or Mexico mint, is a Lamiaceae plant that grows wild in tropical and subtropical
climates. In folk medicine, the decoction of Indian borage leaves was used to treat cough
and skin irritation. Indian borage has also been reported with various pharmacological and
bioactive potentials such as diuretic, antioxidative and antimicrobial properties. The species
of plants that belong to the Lamiaceae family are attributed to high commercial significance.
Plectranthus, Ocimum, Mentha, and Salvia are some of the most important genera in this
family. Ethnobotanical advantages are well-known for these genera. Over 300 species of
Plectranthus have been recognised worldwide (Kumara et al., 2012).

1.8.1. Taxonomy of plectranthus amboinicus

Domain : Eukaryota

Kingdom : Plantae

Phylum : Spermatophyta

Subphylum : Angiospermae

Class : Dicotyledonae

Order : Lamiales

Family : Lamiaceae

Genus : Plectranthus

Species : P. amboinicus (CABI, 2019)

1.8.2. Morphological Features of P.amboinicus

Plectranthus amboinicus is suggested as a succulent shrub with climbing or creeping

habit. The height of its wild forms has been known to exceed 1 m, with a width exceeding 1
m. These have a strong scent and are quite meaty. The stem can grow up to 30-90 cm in
length and has strong hairs (Wagner and Lorence, 2016). The morphology of Leaves is
detailed as simple and quite thick. The leaf is ovate to sub orbicular and has a blunt tip. A
very high number of glandular hairs are seen on the lower surface of the leaves. These hairs
present give a fostered appearance. (Roshan et al., 2010; Kumara et al., 2011). The leaves
have a lovely scent and a good flavour. The flower is a dull purplish colour. A dull purple-
colored corolla which is more than four times bigger than calyx which has a tubular
structure with a short lip is present (Steam, 1992; Roshan et al., 2010).

1.8.3. Origin and Geographical distribution of P.amboinicus

Plectranthus is a Greek word that combines the words "plectron" for spur and "Anthos"
for flower. The presence of spur-shaped blooms in most plants belonging to this genus is
associated with this name (Steam, 1992). Initially, P. amboinicus was categorized in the
genus Coleus and later it was put under the genus Plectranthus, however, both the named
can be seen in literature there are many synonyms used against this species (Morton, 1992).
The plant adapts well in different temperatures when grown in the pot (Steam, 1992).
Organic-rich soil, high humidity, and a neutral pH are essential for optimal P. amboinicus
growth. As a result, this plant grows easily inside and is consequently a common houseplant
in northern Europe.

1.8.4. Nutritional Values

P. amboinicus is a substantial source of chemicals that serve to improve the taste and
shelf life of food. According to the findings, there is a significant concentration of minerals
such as calcium and potassium (Lukhoba et al., 2006). These minerals improve the strength
of bones and optimize the performance of critical organs such as the kidney, heart, nerves,
and muscles, providing significant health benefits. P. amboinicus has a substantial iron
concentration of 0.262 percent. Moreover, the plant also contains zeaxanthin is, neoxanthin,
leptin, violaxanthin, and carotene. Therefore, P. amboinicus can be regarded as a very potent
supplement in diet (Lukhoba et al., 2006; Swamy and Sinniah, 2015).

1.8.5. Uses of P. amboinicus

❖ To increase the taste and aroma of food, P. amboinicus leaves which have a pleasant
aroma is used in cooking. These leaves can also be used as a flavouring agent in
wine and beer (Morton, 1992; Sandhya et al., 2011).
❖ To remove the body’s smell and replace it with fresh smell, the leaves are rubbed on
hairs, hands, and bodies (Morton, 1992; Retief, 2000; Swamy and Sinniah, 2015)
❖ The essential oils from these plants are used for aromatherapy by health workers.
The leaves are mixed with sugar in amazon which behaves as an intoxicant (Prudent
et al., 1995).
❖ In places like Martinique and Tonga, the leaves are used in perfumes and cleaning
textiles (Mohanty et al., 2014; Prudent et al., 1995)

1.9.1. Aim

In this study, the feasibility of Indian borage usage as an ingredient for sustainable
aquaculture of the freshwater prawn, M. rosenbergii was aimed.

1.9.2. Objectives

1. To understand the nursery maintenance of early post larvae of M. rosenbergii.

➢ Under this objective, the survival rate and growth performance were assessed.

2. To check the feasibility of Indian borage as an ingredient in artificial feed formulation for
sustainable growth of M. rosenbergii post larvae.

➢ Under this objective, proximate composition of Indian borage, principle compounds of

secondary phytochemicals of Indian borage leaf powder (both qualitative and quantitative),
survival, growth and nutritional indices were estimated.

3. To see the antioxidant potency of the Indian borage, and its ROS reduction capacity in
Artemia nauplii.

➢ Under this objective, the DPPH free radical scavenging potency and ROS reduction
capacity were evaluated.
2. MATERIALS AND METHODS

Part-I. Nursery maintenance

2.1. Procurement and acclimatization of M. rosenbergii early post larvae (PL)

The culture tanks were cleaned, washed and filled with freshwater and aerated for 24
hours, before leaving the prawns into the tank (Fig.2.1). The early post larvae of M.
rosenbergii (PL, 12) were purchased from Sri Durgai Hatcheries, 120 Ruby Beach Farm,
Paramakeni Village, Chengalpattu, Tamil Nadu, 603305. The early PL was transformed to
the laboratory in well-oxygenated plastic bags and stocked in large cement tank (6’ × 4’ ×
3’) for acclimatization (Fig.2.2).

About 80% of water was routinely changed every day in order to maintain a healthy
environment for the PL apart from providing artificial aeration. This ensures sufficient
oxygen supply for PL and an environment devoid of accumulated metabolic wastes. The
unfed feed, feces, moult and dead prawns (if any) were removed by siphoning without
disturbing the prawns. During acclimatization, the early PL was fed eight times per day
(6.00 a.m., 8.00 a.m., 11.00 a.m., 1.00 p.m., 3.00 p.m., 5.00 p.m., 7.00 p.m., 10.00 p.m.,)
for 28 days. The first and last feeding of a day was with boiled egg albumin (two times per
day), and the remaining feeding was with live Artemia nauplii (six times per day).

During the period of nursery maintenance, the following range of water quality parameters
were estimated. The dissolved oxygen was between 7-8 mg/L (measured by DO meter).
The pH was in the range of 7-7.3 (measured by pen type pH meter). Salinity was estimated
between 0.6-0.8 ppt (measured by pen type salinometer). The ammonia level was between
0.5- <1.0 (measured by a kit method).
Fig.2.1. Preparation of culture tanks Fig. 2.2. Acclimatization of prawn to
the laboratory environment

2.1.2. Determination of growth performance of M. rosenbergii, early PL

M. rosenbergii early post larvae ranging from 1.5±0.1 cm in length and 0.04±0.01 g in
weight were used for the determination of nutritional indices and the final day PL was used
as the initial day PL for the further experimental procedures.

Nutritional indices, such as survival rate (SR), length, length gain (LG), weight, weight
gain (WG) and specific growth rate (SGR) were individually determined by following
equations (Terkinay & Davis, 2001).

Survival (%) = Total number of live animals / Total number of initial animals × 100

Mortality rate (%) = Total number of animals died/ Total number of initial animals × 100

Cannibalism rate (%) = (Survival rate + Mortality rate) -100

Length gain (cm) = Final length (cm) – Initial length (cm)

Weight gain (g) = Final weight (g) – Initial weight (g)

Specific growth rate (%) = log W2 – log W1/t × 100

Where,

W1 & W2 = Initial and Final weight respectively (g), and t = Total number of experimental
days.

Fig.2.3. Initial day early PL of M. Fig.2.4. Final day early PL of M.

rosenbergii in nursery maintenance rosenbergii in nursery maintenance
(initial day PL in feeding trial)

Part-II. Indian borage as a feed ingredient

2.2. Collection and Processing of Indian borage leaves

The medicinal herb Indian borage, Plectranthus amboinicus was collected, from the
area of Thudiyaloor at Coimbatore, Tamilnadu, India. They were collected and washed
with water to remove the dust particles and dried at room temperature for about 2-3 days.
Later it was completely dried and devoid of moister content, the leaves were grinded to a
fine powder, sieved using a fine mesh strainer and stored in sterile container.
Fig.2.5. Indian borage leaves collected Fig.2.6. Dried leaves of Indian borage

2.3. Analysis of Proximate Composition of Indian borage leaf powder

The proximate composition of Indian borage, such as, moisture, crude protein, crude
fiber, etheric extract, total ash, total nitrogen free extract, acid insoluble ash, calcium,
phosphorus, salt and gross energy of Indian borage (P.amboinicus) were analyzed by
adopting outsourcing service at Tamil Nadu Veterinary College and Research Institute
(TANUVAS), Namakkal, Tamil Nadu, India), the detailed procedures are given in
Annexure 1.

2.4. Preparation of extracts (Soxhlet extraction and Rotary evaporation)

The powdered sample, P.amboinicus leaf was weighed (75g) and packed in Whatman
No.1 filter paper. The solvent (500 ml of methanol) is added to a round bottom flask,
which is attached to a Soxhlet extractor and condenser on an isomantle. The powdered
plant material is loaded inside the Soxhlet extractor. It was sequentially extracted with
methanol for 6-9 hours each (30-36 cycles) using solvents based on their polarity (non-
polar to polar). Repeated extraction was done until a clear colourless solution was
obtained. The extracts were filtered by using double layer muslin cloth, and there were
concentrated at 40-50oC using rotary vacuum evaporator (ROTAVAP). The extract
obtained were vacuum dried under 40oC and used for further investigation.

The aqueous extract of P.amboinicus grain powder was also taken and subjected to
qualitative and quantitative estimations of the principal secondary phytochemicals.

Fig.2.7. Methanolic Extract of Plectranthus amboinicus

Fig.2.7. Methanolic Extract of Plectranthus amboinicus after

condensation in rotary evaporation

2.5. Analysis of the Principle Secondary Phytochemicals of the methanolic extract of

P.amboinicus leaves
The principle secondary phytochemical analysis of the methanolic extract of
P.amboinicus was analyzed for the presence or absence of the principle secondary
phytochemicals such as, phenols, tannins, flavonoids, saponins and alkaloids were
analyzed qualitatively and for the analysis of the quantity of the compounds, phenols,
tannins and flavonoids were estimated quantitatively. The detailed procedures are given in
Annexure II.

2.6. Feed preparation

The feed ingredients were purchased from the local shops at Coimbatore. There were
mixed and prepared by grinding the feed ingredients, mixing, steam cooking and
pelletization of the feed and checking the quality of the prepared feed.

Grinding

The basal ingredients, such as fishmeal, soybean meal, wheat bran, tapioca flour and
groundnut oil cake were ground separately using a micro pulverizer

Mixing

Powdered feed ingredients were weighed out to prepare feed mix based on Pearson
Square method equated to 40% protein.

Steam cooking

The feed mixes along with Indian borage leaf powder (3%, 5% and 7% was steam
cooked for 5 minutes at 95-100oC and allowed to cool at room temperature. Groundnut
oil, egg albumin and vitamin B-complex (Becosules, Pfizer) were also thoroughly mixed.

Pelletization and quality check

The feed was pelletized in a manual pelletizer fixed with 3 mm diameter die and pellets
were collected in aluminum trays. Then the feed was shadow dried until the moisture
content is less than 10%. The shadow dried feed pellets were physically examined for
visual appearance, such as uniformity, color and fragrant smell. The pellets were prepared
with smooth surface. These feeds were fed to the PL under different experimental setup
for analysis of different parameters.
The proportion of the basal ingredients and the incorporation of Indian borage leaf powder
was prepared as feed for the freshwater prawn, M. rosenbergii PL in the following ratio.

Experimental diets:

• Basal ingredients + 3% of Indian borage leaf powder incorporated artificial

diet.
• Basal ingredients + 5% of Indian borage leaf powder incorporated artificial
diet.
• Basal ingredients + 7% of Indian borage leaf powder incorporated artificial
diet.

Table 2.1. Proportion of basal ingredients used in control diet

Control diet:
Basal ingredients Ratio (g) **Each capsule contains,
Soybean meal 25 Thiamine Mononitrate IP 10 mg
Fish meal 25 Calcium pantothenate IP 50 g
Groundnut oilcake 25 Riboflavin IP 10 mg
Wheat flour 10 Folic acid IP 1.5 mg
Tapioca flour 5 Pyridoxine Hydrochloride IP 3 mg
Egg albumin 7 Biotin USP IP 100 mcg

Groundnut oil 2 Vitamin B12 (as tables 1:100)IP 15 mcg

Vitamin mix** 1 Ascorbic acid IP 150 mg

Niacinamide IP 100 mg

** BECOSULES CAPSULES Manufactured by Pfizer

Fig. (i). Control diet Fig. (ii). 3% of Indian borage powder
incorporated artificial diet

Fig. (iii).5% of Indian borage leaf Fig. (iv). 7% of Indian borage leaf
powder incorporated artificial diet powder incorporated artificial diet
2.6.1. Feeding trial

The freshwater prawn, M. rosenbergii post larvae ranging from 1.5±0.1 cm in length
and 0.04±0.01 g in weight respectively were used for the feeding experiment. For each
diet, triplicate was maintained in plastic troughs with 20 L water. 1. Basal diet without the
addition of Indian borage served as control. 2. Basal diet with the incorporation of Indian
borage in four different concentrations (3%, 5% and 7%). The feeding was adjusted to two
times a day (6.00 am and 6.00 pm). The daily ration was given at the rate of 10% of the
body weight of PL with two equal halves throughout the experimental period. The
experimental period was prolonged for 45 days; mild aeration was given continuously in
order to maintain the optimal oxygen level.

Fig.2.9. M. rosenbergii post larvae of initial day feeding trial

(Final day early PL of nursery maintenance)

2.6.2. Survival, growth and nutritional indices

Nutritional indices such as, survival rate, mortality rate, length gain, weight gain,
specific growth rate, feed conversion ratio was calculated as follows (Terkinay & Davis,
2001).

Survival rate (%) = Final prawn number / Initial prawn number×100

Length gain (%) = Final length – Initial length (cm)

Weight gain (%) = Final weight – Initial weight (g)

Specific growth rate, (%) = log W2 – log W1/t × 100

Where,

W1 & W2 = Initial and Final weight respectively (g), and t = Total number of
experimental days.

Feed conversion ratio (%) = Total feed consumed (dry weight) g / Live weight gain (wet
weight) g

Part III. Free radicals scavenging property of Indian borage

2.7. In vitro antioxidant activity of Indian borage leaf powder extract

2.7.1. DPPH radical scavenging assay

The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the medicinal

herb, Plectranthus amboinicus was measured according to the protocol adopted by Dore et
al., (2015). Different concentrations (3%, 5% and 7%) of plant extract were prepared and
subjected to antioxidant tests. Briefly, 0.1ml of plant extract from final concentration of
the solution was added with 0.9 ml distilled water and 3 ml of 0.1 mM DPPH in methanol
to make for 4 ml, followed by incubation at 27oC, for 30 minutes. After incubation, the
absorbance was measured by using a spectrophotometer at 517 nm.

The percentage inhibition activity was calculated using the formula:

Scavenging activity (%) = [(A0 - A1) / A0] × 100

Where A0 is the absorbance of control, and A1 is the absorbance of the extract/standard.

Ascorbic acid was used for positive control and methanol used as negative control. The
mean values were obtained from three independent experiments.

2.8. ROS Reduction capacity of Indian borage leaf powder in Artemia nauplii

2.8.1. Enrichment
The enrichment techniques were employed to improve the dietary value of Artemia
franciscana.

Enrichment of Artemia franciscana nauplii using P.amboinicus leaf powder

The brine shrimp, Artemia franciscana cyst was purchased from Aqua world, Paris
Corner, Chennai, India. The cysts (2g/20 L) were taken and hydrated in 1 L of purified
artificial saltwater (prepared from artificial sea salt powder) for 12-15 hours. The cysts
burst and the embryos surround by the hatching membrane become visible for few hours.
The brownish orange colour indicates the hatching of the nauplii. Artemia nauplii were
filtered and transferred to 1 L capacity glass beaker. Three such groups were enriched
with 3%, 5% and 7% concentrations of plant extract for one hour.

2.8.2. Intracellular ROS generation in Artemia nauplii

Total ROS levels were quantified in whole Artemia nauplii using 2’, 7’-
dichlorodihydrofluorescin-diacetate (H2DCFDA), (Sigma-Aldrich). ROS levels were
determined according to previously performed protocols with slight modifications
(Rekulapally et al., 2019). Briefly, Artemia were fed with different concentrations (3%,
5% and 7%) plant extract for 2 hours. Plant extract was removed by washing three times
with PBS buffer, and Artemia were suspended in PBS buffer. 3 ml aliquot of the
suspension was transferred into each well of a black 12-well plate, and a 50-µL aliquot of
the freshly prepared 100 µM, H2DCFDA solution was added, resulting in a final
concentration of 50 µM. These were incubated for 30 minutes, and then treated Artemia
were mounted on 75% glycerol for examination with a fluorescence microscope. The
fluorescent intensity of the intestine was quantified using “Image J” software and
expressed as relative fluorescent unit (RFU) u. Artemia were examined per treatment with
three independent biological replicates.
3. RESULTS

Part-I. Nursery maintenance

3.1. Nursery Maintenance of M. rosenbergii early PL

3.1.1. Survival and Growth performance

The morphometric data, length and weight, and nutritional indices, such as Survival
Rate, Weight Gain and Specific Growth Rate were found to be obviously increased from
the initial day of early PL to the final day of the early PL (Table 3.0).

Table 3.0. Survival and growth parameters of M. rosenbergii early PL during nursery
maintenance

Parameters Nursery Maintenance

Initial length (cm) 0.9±0.08
Final length (cm) 1.5±0.1
Initial weight (g) 0.02±0.01
Final weight (g) 0.04±0.01
Length gain (cm) 0.56±0.11
Weight gain (g) 0.02±0.01
Specific growth rate (%) 3.1275±0.38
Survival rate (%) 50%
Mortality rate (%) 35.62%
Cannibalisms rate (%) 14.38

Each value is mean ± SD of five individual observations.

Part-II. Indian borage as a feed ingredient

3.2. Proximate composition of Indian borage leaf powder

The proximate composition, such as, moisture, crude protein, crude fiber, etheric
extract, total ash, total nitrogen free extract, acid insoluble ash, calcium, phosphorus, salt
and gross energy of Indian borage leaf powder is displayed in table 3.1. The Indian
borage leaf powder contains 9.72% moisture, 10.91% crude protein, 10.06% crude fiber,
3.26% Ether extract, 19.27% total nitrogen free extract, 0% total ash, 0.62% acid insoluble
ash, 3.20% calcium, 0.28% phosphorus, 3.0% salt and 3198 kcal/kg gross energy.

Table 3.1. Proximate composition of Indian borage (P.amboinicus) leaf powder

Parameters Method Composition

Moisture (%) AOAC (1995) 9.72
Crude Protein (%) AOAC (1995) 10.91
Crude Fibre (%) AOAC (1995) 10.06
Ether Extract (crude fat) (%) AOAC (1995) 3.26
Total Nitrogen Free Extract (%) Castell & Tiews (1980) 19.27
Total Ash (%) AOAC (1995)
Acid Insoluble Ash (sand & silica) (%) AOAC (1995) 0.62
Calcium (%) AOAC (1995) 3.20
Phosphorus (%) AOAC (1995) 0.28
Salt (%) AOAC (1995) 3.0
Gross Energy (kcal/kg) AOAC (1995) 3198

3.3. The principle secondary phytochemicals of the methanolic extract of Indian

borage leaf powder
The methanolic extract Indian borage leaf powder is screened for the principle
phytochemical analysis (qualitative and quantitative) (Fig. 3.1& 3.2) for phenols, tannins,
flavonoids, saponins and alkaloids of which, phenols, flavonoids and alkaloids were found
to be luxuriantly present, Tannin were moderately present and saponins were poorly
present in P.amboinicus (methanolic extract). Similarly, phenols, tannins, flavonoids and
alkoloids were found to be luxuriantly present and saponins, were found to be absent (in
aqueous extract). (Table 3.2 & 3.3).
Table 3.2. Qualitative analysis of principle secondary phytochemicals present in the
methanolic extract of P.amboinicus leaf powder

Phytochemicals Methanolic extract of Aqueous extract of

P.amboinicus leaf powder P.amboinicus leaf
powder

Phenol +++ +++

Tannins ++ +++
Flavonoids +++ +++
Saponins + --
Alkaloids +++ +++
+
poorly present; ++moderately present; +++luxuriantly present; --Absent

Table 3.3. Quantitative analysis for principle secondary phytochemicals present in

the methanolic extract of P.amboinicus leaf powder

Phytochemicals Methanolic extract of Aqueous extract of

P.amboinicus leaf powder P.amboinicus leaf
powder

Phenol 4.433± 0.0029 4.434± 0.0017

Tannins 4.1037 ± 0.0017 4.1267 ± 0.002
Flavonoids 0.5447 ± 0.002 1.357 ± 0.0021

Each value is mean ± SD of three individual observations.

Values between aqueous and methanolic extract of P.amboinicus leaf powder were
significant at p<0.05.

3.4. Survival, Growth and Nutritional Indices

Nutritional indices parameters such as survival rate, length gain, weight gain, specific
growth rate, feed conversion ratio is given in table 3.4. Survival rate was significantly
higher in prawns fed with Indian borage leaf powder supplemented diets fed PL groups
when compared with control. The length gain, weight gain, specific growth rate was
significantly higher in 5% diet fed PL group, followed by 7%, 3% when compared with
control. The feed conversion ratio was significantly lower in 7% Indian borage
incorporated diet fed PL groups followed by 5 and 3% when compared with control.
(Table 3.4).

Table 3.4. Nutritional indices of M.rosenbergii PL fed with different concentrations of

Indian borage leaf powder incorporated diets

Parameter BI (Control) Indian borage leaf powder incorporated diets

BI + 3% BI + 5% BI + 7%
43.33±4.71 56.67±4.72 73.33±9.43 53.33±9.43
SR (%)
1.61±0.042 1.84±0.14 2.58±0.04 2.05±0.01
Length(cm)
0.11±0.42 0.34±0.14 1.08±0.04 0.55±0.01
Weight (g)
0.08±0.001 0.09±0.12 0.16±0.02 0.15±0.01
WG (g)

SGR (%) 2.004±0.007 2.08±0.003 2.33±0.05 2.27±0.04

FCR (g) 1.37±0.14 1.23±0.11 0.69±0.05 0.57±0.01

BI, basal ingredients

Each value is mean ± SD of three individual observations.
Values between control and methanolic extract of P.amboinicus leaf powder were
significant at p<0.05
Fig (i). Post larvae of M. rosenbergii fed with Fig (ii). Post larvae of M. rosenbergii fed with
Control diet 3% of Indian borage leaf powder incorporated
artificial diet

Fig (iii). Post larvae of M. rosenbergii fed with Fig (iv). Post larvae of M. rosenbergii fed with
5% of Indian borage leaf powder incorporated 7% of Indian borage powder incorporated
artificial diet artificial diet

Fig.3.1. M. rosenbergii PL, fed with different concentrations of Indian borage leaf
powder incorporated diet

Part III. Free radicals scavenging property of Indian borage

3.5. DPPH free radical scavenging activity of P.amboinicus leaf powder

Antioxidants are the organic substances that are highly utilized through natural
sources which is also a combination of complex phytocompounds. Due to extreme
generation of oxidative stress by pro oxidants, a condition is developed where the cellular
molecules such as proteins, lipids, and nucleic acids suffer oxidative damages and may
cause tissue disruption (Halliwell & Aruoma, 1991; Land, 1990). The radical
scavenging activities of different concentration (3%, 5% and 7%) of P.amboinicus leaf
powder were analyzed with reference to 2, 2-diphenyl-1-picrylhydrazyl (DPPH), a stable
free radical. The concentration of P.amboinicus at 7% exhibited maximum DPPH
scavenging activity of 60.34% followed by 5% (50.44%) and 3% (25.2%) (Fig.3.2). The
data revealed that the DPPH free radical scavenging activity of P.amboinicus was
dependent on the concentration. The strong antioxidant activity of plant extract suggests
that it can be used for production of ethano-medicine activities without any cytotoxicity or
adverse side effects. P.amboinicus exhibited strong free radical scavenging activity (Fig.
3.2).

Table 3.6. DPPH free radical scavenging activity of Indian borage powder

Concentration of Indian OD of DPPH free radicals

borage leaf powder
Control 1.09±0.01
3% 0.82±0.07
5% 0.54±0.007
7% 0.43±0.03

Each value is mean ± SD of four individual observations

Fig.3.2. DPPH free radical scavenging activity (%) of P.amboinicus leaf powder

3.6. Enrichment of Artemia nauplii with P.amboinicus leaf powder

Artemia are widely used as live prey in aquaculture. The lack of n-3 highly unsaturated
fatty acids (HUFA), essential for the aquatic organisms, makes it necessary to enrich
Artemia nauplii in order to improve the healthy profile. In the present study, Artemia
franciscana nauplii were used for the enrichment. The Artemia nauplii was enriched with
the leaf powder P.amboinicus, where it showed significant results. The feed intake was
found to be increased in 7% followed by 5% and 3%. The unenriched Artemia nauplii
served as control.
Fig (i). Control (unenriched Artemia Fig (ii). Artemia nauplii enriched with 3%
nauplii) concentration of P.amboinicus

Fig (iii). Artemia nauplii enriched with Fig (iv). Artemia nauplii enriched with 7%
5% concentration of P.amboinicus concentration of P.amboinicus

Fig.3.3. Artemia Enrichment with P.amboinicus leaf powder at three different

concentrations (3%, 5% and 7%)
Control (un-enriched, but ate other food)

Fig.3.4. Relative enrichment of Artemia nauplii (Mean RFU)

3.7. ROS reduction capacity of Indian borage leaf powder in Artemia nauplii

Inside cells, H2DCF-DA is subjected to oxidation in the presence of intracellular

ROS. The oxidized compound emits fluorescence, the intensity of which correlates with
intracellular ROS levels. As showed in (Fig.3.5) P.amboinicus at different concentrations
(3%, 5% and 7%) significantly reduced the intracellular ROS levels in treated Artemia
nauplii to respectively when compared to untreated Artemia nauplii. The reduction in ROS
levels can be assumed to be the chief reason behind stress resistance in Artemia nauplii.
P.amboinicus leaf powder reduced the production of the ROS generation based on the
concentration.

Fig (i). Control (unenriched Artemia Fig (ii). Artemia nauplii enriched with 3%
nauplii) of P.amboinicus
Fig (iii). Artemia nauplii enriched with 5% Fig (iv). Artemia nauplii enriched with 7%
of P.amboinicus of P.amboinicus

Fig.3.5. Reduction of ROS production in Artemia (based on the green fluorescence

color intensity reduction)

Fig.3.6. Relative ROS production in Artemia (Mean RFU)

4. DISCUSSION

4.1. Growth and Survival rate (in nursery maintenance)

In aquaculture, growth is an index of water quality and nutrition in a pond. Growth

of prawn is normally very fast during the early life and slows down in adults; the survival
rates are also very high during the early life and fell subsequently (Ling, 1967). The slow
growth rate is accompanied with oxygen depletion, food scarcity and competition.
Aquaculture with compatible species and good farm management generally yield normal
growth.

In the present study, the early post larvae were fed two feeds, egg two times per
day, and Artemia nauplii six times per day. It showed a significant growth in the early post
larvae and found to increase the growth and survival of the early post larvae.

4.2. Proximate composition of Indian borage leaf powder

Indian borage has become popular as a traditional medicinal food due to its high
mineral and antioxidant content. Its benefits may include blood purification, to increase
urine flow and to prevent inflammation of the lungs. The nutritional value of Indian
borage is considerably higher than that of many other medicinal plant.

4.2.1. Protein and carbohydrates

Protein is important for growth and maintenance of body tissue. It provides energy
and is needed for the production of hormones. Indian borage has a well-balanced amino
acid profile, because of that, the protein in Indian borage is considered as a very high-
quality protein. It is rich in the amino acids, lysine and arginine. In animals, Indian borage
protein has proven treating at indigestion, improving appetite, and disorder of digestive
system, and reducing the risk of colon cancer.

4.2.2. Fiber and fatty acids

Fiber is a type of carbohydrate that the body can’t digest. Indian borage contains a large
amount of fiber. This nutrient is good for colon health. Indian borage contains 10.06% of
crude fiber and is mainly composed of lignin and cellulose. It has resistant starch, which is
resistant to digestion and is thus categorized as fiber. Resistant starch is fermented by gut
bacteria in the colon. Fiber helps regulate the body’s use of sugars, helping to keep
appetite and blood sugar in check.

4.3. Formulated Diets

Feed is the major operational cost for most aquaculture enterprises (Abramo and Sheen,
1994). The most significant aspects of successful aquaculture are the creation of well-
balanced diets and their proper feeding. Animal and plant by-products play an important
part in artificial feed composition. Plant feed stuffs created in aqua feeds for the production
of aquatic species, in particular, are a crucial prerequisite for aquaculture's future
development. Such plant feedstuffs must provide cost-effective diets that will allow
aquatic animals to develop with minimal environmental impact while also producing a
tasty and healthy product. (Vista, 2008).

4.4. Growth and Survival rate (in experimental PL)

In this study, the survival and growth performance of M. rosenbergii were higher in Indian
borage incorporated diet fed PL groups when compared with control. It has been reported
that the Indian borage leaf powder incorporated diet fed to PL of M. rosenbergii had
improved the survival and growth performance of M. rosenbergii.

4.5. DPPH Radical Scavenging activity

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay

based on electron-transfer that produces a violet solution in ethanol. This free radical,
stable at room temperature, is reduced in the presence of an antioxidant molecule giving
rise to colorless ethanol solution. Due to antioxidant activity of naturally occurring
substances in higher plants, attention has increased on the protective activity of these
natural antioxidants against chronic disorders caused by oxidative stress. The use of the
DPPH assay provides an easy and rapid way to evaluate antioxidants by spectrophotometry
(Huang DJ, et al., 2005). In the present study, P.amboinicus has resulted in showing high
antioxidant activity. The DPPH scavenging activity of Indian borage has increased when
the concentration increases. At 3% concentration, it showed 25.2% DPPH scavenging
activity and when it is increased to 5% and 7%, the DPPH Scavenging activity was
increased to 50.44% and 60.34% respectively.

4.6. ROS (Reactive Oxygen Species)

Harman’s free radical theory of aging (FRTA) highlight the role of mitochondria in aging
process, since mitochondria are considered as the main source of ROS generation
(Harman, 1972). Because there are many ROS, that are not free radicals and that can also
cause oxidative damage. The FRTA is now referred as the oxidative stress theory of aging
(Sohl and Weindruch, 1996). This new name for the theory also implies that oxidative
stress can occur due to an imbalance between ROS production and removal. The present
study assessed the effects of Indian borage leaf powder at different concentration on ROS
accumulation, the cell permeable reagent H2DCF-DA (2′, 7′-dichlorofluorescin-diacetate)
was employed. This compound becomes deacetylated by intracellular esterases and
remains inside the cells. Inside cells, H2DCF-DA is subjected to oxidation in the presence
of intracellular ROS. The oxidized compound emits fluorescence, the intensity of which
correlates with intracellular ROS levels. P.amboinicus at different concentrations (3%, 5%
and 7%) significantly reduced the intracellular ROS levels in treated Artemia nauplii to 64,
48 and 34 percent respectively when compared to untreated Artemia nauplii. The reduction
in ROS levels can be assumed to be the chief reason behind stress resistance in Artemia
nauplii.
5. SUMMARY AND CONCLUSION

Aquaculture of freshwater prawns earns valuable foreign exchange. The giant river prawn,
Macrobrachium rosenbergii culture is much preferable and profitable. The culture of M.
rosenbergii generates considerable employment opportunities. However, the farming cost
would be non-affordable to small farmers. Among various sectors, the feed accounts for
60% of total operational cost. The freshwater prawn farming is currently threatened by
low production efficiency. The major constraints in this industry are non-availability of
healthy seed supply and culture techniques. In addition, the degradation of natural habitats
caused great threats to freshwater giant prawn populations. Therefore, development of
more sustainable and efficient prawn production system is required. This can be achieved
through supplementation of good quality feed for nutrient utilization, general health
promotion, water quality maintenance, stress resistance and disease tolerance in M.
rosenbergii.

In the present study, two different feeds, egg albumin two times per day and live artemia
nauplii six times per day were given as feed for the freshwater prawn, M. rosenbergii early
PL, for 28 days in nursery maintenance. It was found to produce better survival and
growth performance of the early PL, in which 50% survival and the length gain was
1.5±0.1 cm and the weight gain was observed as 0.04±0.01g. The growth performance has
increased from the initial day of early PL.

The formulated diet with Indian borage powder was given as a feed for the freshwater
prawn, M. rosenbergii, during the experimental period for 45 days. Investigation on the
proximate composition of Indian borage powder showed the presence of high-quantity
protein content in it. And it showed a great range of carbohydrates, moisture content and
lipid in it. The 5 principle secondary phytochemicals, such as phenols, tannins, flavonoids,
saponins and alkaloids were found to be present in Indian borage methanolic extract. Of
which, the phenols, tannins flavonoids and alkoloids were found to be luxuriantly present
when quantified and saponins were found to be absent.

Indian borage powder incorporated feed was given to the PL, where it showed better
survival and nutritional indices. Differences in nutritional indices were observed between
the control and the experimental groups and within the experimental groups. When
compared with control, the produced better growth performance was in the order of 5% >
7% > 3%. Indian borage showed some antioxidant potency and it exhibited reduced ROS
production in Artemia.

Recommendation

In the present study, the overall performance of Indian borage leaf powder incorporated
diets on survival, growth and production of M. rosenbergii PL were appreciable.
Therefore, Indian borage leaf powder can be taken as an ingredient in feed formulation for
promoting sustainable aquaculture of freshwater prawns.
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7.1. Annexure-I

Proximate composition analysis of buckwheat

The proximate composition of Indian borage Plectranthus amboinicus, such as crude

protein, crude fiber, ether extract, moisture, ash, gross energy was analyzed by adopting
the following methodologies.

1. Determination of moisture content

The moisture content was determined by dry sample in hot air oven as described in AOAC
(1995). 5 g of feed sample was taken on pre-weighed concave glass and they were kept in
desiccators, to maintain 0.5% relative humidity. The dried sample was kept in the
desiccators at (105oC for 24 hours) until they reach at a constant weight.

The moisture content of the samples was calculated as,

2. Crude protein analysis (Kjeldahl Method) (AOAC, 1995)

Principle: Sample was digested in H2SO4 by using CuSO4 as catalyst, N converted to NH3
which was distilled and titrated.

Reagents

1. Sulfuric acid: Specific gravity 1.84, N-free.

2. Mercuric oxide: N-free.

3. Potassium sulfate: N-free.

4. Sodium hydroxide-sodium thiosulphate solution: 60g of NaOH and 5g

Na2S2O3.5H2O was diluted in 100 ml of H2O.

5. Boric acid solution: Saturated solution.

6. Indicator solution:
Methyl red-methylene blue: 2 parts 0.2% alcoholic methyl red solution was mixed
with 1 part 0.2% alcoholic methylene blue solution.

Methyl red-bromocresol green solution: 1 part 0.2% alcoholic methyl red solution
was mixed with 5 parts 0.2% alcoholic bromocresol green solution.

7. Hydrochloric acid: 0.02N.

A. Apparatus

B. Digestion rack: With either gas or electric heaters supply heat to 30 ml flask to
cause 15 ml of H2O at 25oC to boil at < 3 minutes.

1) Distillation: The digestion flask was connected to distillation trap by a rubber

stopper. Distillation trap was connected to condenser at low-S tubing. Outlet of
condenser tube was fixed as < 4 mm diameter.

2) Digestion flasks: 30 ml regular Kjeldahl or Soltys-type flask was taken. For

small samples 10 ml Kjeldahl flasks were used.

C. Determination

5 ml of 0.01 N HCl was taken and transferred to 30 ml of digestion flask. The charging
tubes used for dry solids, porcelain boat for sticky solids or non-volatile liquid and
capillary or capsule for volatile liquids. To the 2g of K2SO4, 40 mg of HgO and 2 ml of
H2SO4 were added. To the 15 mg of sample and 0.1 ml of H2SO4 was added in each 10 mg
dry organic matter. Boiling chips pass through No. 10 sieve. The boiling time for
digestion rack heater was 2 minutes digested for one hour and the D. H2O was added until
the acid comes to true boil; the boiling time was noted for 3 minutes and digested for 1.5
hour. The minimum value of H2O was cooled and added to dissolve solids. The digested
and boiled chips were transferred to distillation apparatus and the flask was rinsed with 5
times with 2 ml of H2O to the 5 ml of saturated H3BO3 solution 4 drops of indicator was
added under condenser with tip extending below surface of solution to 10 ml of NaOH-
Na2S2O3 solution, 15 ml of Ca was distillated and diluted to 50 ml of Ca (to 2.5 ml H 3BO3
add 2 drops of indicator was diluted with 25 ml of Ca along with 0.01 N HCl). Titration
was done till the end point.
Calculation

The crude protein content of the sample was calculated as:

3. Crude fiber analysis

The crude fiber of the feed sample was estimated as per AOAC (1995). Feed sample (5 g)
was digested with 0.128 M H2SO4 with 2 drops of octanol in digestion unit (Hot extractor,
Model-1017) for 30 minutes. The acid in the sample was removed by filtered and washed
with hot water, further residue was boiled with 0.233 M KOH for 30 minutes, again rinsed
with boiling water and acetone. The residue was dried in an oven at 130 °C for 2 hours and
ignited in muffle furnace at 500 °C for 3 hours. The loss of weight represented the crude
fiber content in the sample.

The crude fiber content of the sample was calculated as:

Where

• A = weight of crucible with dry residue (g)

• B = weight of crucible with ash (g)
• C = weight of sample (g)

4. Etheric extract or crude fat analysis (AOAC, 1995)

Crude fat content of moisture free feed sample was determined by extracting the fat with
petroleum ether by using Soxhlet apparatus as described in AOAC (1995). Briefly, 10 g
of moisture free feed sample was taken in an extraction thimble and it was placed in the
extractor with an attachment receiving flask. The solvent was poured into the thimble
through a glass funnel. The receiver containing petroleum ether was heated (60 °C) at such
a rate the ether drops from the condenser to the thimble at the rate of 5 drops per second.
When sufficient solvent was transferred to the extracting tubes to fill the siphon arm, it
siphoned into the receiver. This process was continued until the extraction gets completed
(around 17 hours). After that, the flask was removed and the volatile solvent was
evaporated at 80 °C on a rotary flash evaporator. The residue was dried in an oven and
cooled in a desiccator and weighed. The least weight of residue gives the weight of fat in
the sample. The fat content of the sample was expressed on wet weight basis as percentage.

The crude fat content of the sample was calculated as:

Crude Fat (%) = (W3 - W2) × 100 / W1 × Lab DM / 100

Where,

• W1 = initial sample weight in grams

• W2 = tare weight of beaker in grams
• W3 = weight of beaker and fat residue in grams

5. Determination of total ash

Principle

Principle involved that, when a known weight of feed was ignited to ash, the weight of ash
thus obtained was expressed in terms of percentage.

Apparatus

• Silica crucible

• Tongs

• Weighing balance

• Electrical Bunsen burner

• Muffle furnace

• Desiccator

• Asbestos sheet

Procedure

1. The clean dry crucible was taken and weighed.

2. 2 g of sample was taken and weighed to get accurate weight of the sample.
3. The weighed crucible was placed on the electric burner. The crucible was partially
opened.

4. The charred sample was noted with initial expulsion of smoke.

5. The crucible muffle furnace was heated to 600oC. It was kept for 2 hours. All organic
matters were burnt leaving the minerals at this temperature.

6. The crucible was removed from the furnace carefully and cooled it in a desiccator at
room temperature and weighed again.

Calculation

Ash content (%) - (Z – X / Y – X) × 100

Weight of empty crucible - Xg

Weight of crucible + sample - Yg

After complete ashing, Weight of crucible + ash - Zg

The weight of the ash sample was taken after the complete combustion of total ash. It was
comprised with two portions. The soluble portion in diluted acid contains all essential
minerals and useful portion of ash. The other portions contained insoluble in diluted acid
consists of mainly sand and silica. For the most part it was represented as impurity or
adulteration.

6. Estimation of Gross energy

Gross energy was calculated by using the energy values contributed by the protein, NFE
(Nitrogen-Free Extract) and lipid fractions of feed by multiplying generalized
physiological energy values of proteins (19 kJ), NFE (15 kJ) and lipids (36 kJ)
respectively, the detailed calculations was followed by Natarajan (2006).

Energy contributed by crude protein (kJ/g) = protein content of feed (%) × 19 = x

Energy contributed by crude lipid (kJ/g = lipid content of feed (%) × 36 = y

Energy contributed by NFE (kJ/g) = NFE content of feed (%) × 15 = z

Gross energy (GE kJ/g) = (x + y + z) / 100

7. Determination of Acid Insoluble Ash (Sand & Silica)

Reagent

A. 2N Hydrochloric acid (HCl)

1. To 166.7 ml of concentrated HCl and 700 ml of D. H2O was taken and mixed well.

2. Stirred and prepared to 1 L.

Procedure

A. 5 g of sample was taken in duplicate and tared as 50 ml in ash crucible.

B. The sample was dried overnight at 100oC. The crucibles were allowed to cool in desicator
and reweighted.

C. Ash remained for 6 hours at 600oC.

D. Ash was transferred to 600 ml Berzelius beaker by adding 100 ml of 2 N HCl.

E. It was boiled 5 minutes on fiber rack.

F. Filter hot was hydrolyzed through Whatmann 541µm filter paper and washed with hot
distilled water.

G. The filter paper was transferred back into crucible and the ash remained 6 hours at 600oC.

H. Crucible was placed at 100oC in oven to re-dry. The desiccator was cooled and weighed.

Calculation

Acid Insoluble ash = (Wt. of Crucible + Ash – Wt. of Crucible) × 100

_______________________________________
Sample Dry Weight

8. Determination of calcium

Five dry beakers were taken and 250 ml of deionized water was added. 500 µL automatic
pipettes was taken and added as 0.0, 0.5, 1.0, 1.5 and 2.0 ml of the calcium stock solution
were added to the beakers and mixed thoroughly. The concentrations of calcium in these
standards were calculated.

The standards and unknown samples were measured before switching to another element.
The unknown sample were diluted and the absorbance was measured.

9. Determination of phosphorus

2 g of ash sample was taken in 150 ml beaker for about 4 hr at 600oC. 40 ml of HCl and
several drops of HNO3 was added and transferred to 200 ml volumetric flask then diluted
to volume with H2O. The aliquot contained 1.5 mg of P 100 ml volumetric flask. 20 ml
molybdovanadate reagent was added and diluted to volume with H2O and mixed well for
10 minutes. Then read percent T at 400 nm against 0.5 mg of standard was set at 100% T
(15 mm diameter cells). The determined mg P was followed from standard curve.

Calculation

P (%) = mg P in aliquot / (g sample in aliquot × 10)

10. Determination of Salt (AOAC, 1995)

Preparation

The feed sample taken (≤ 100g) and passed for 3 times through food mincer or chopped
finely and mixed thoroughly.

Reagents

All reagents were from GR grade or AR grade

a) 0.1N silver nitrate (AgNO3) solution: 17g of AgNO3 was dissolved in distilled water and
made to 1 liter in volumetric flask. Later it was stored in a brown color glass bottle in the
dark.

b) Potassium chromate indicator (K2CrO4): 5g of K2CrO4 was dissolved in 100 ml distilled

water.

Procedure

1. 25 g of sample was taken in 400 ml beaker.

2. 200 ml of hot boiled water was added and stirred for 60 minutes.

3. Sample was filtered through the glass wool. Filtered sample was collected in a 250 ml
volumetric flask. It was made up to the volume and mixed well.

4. 10 ml of filtrate was taken in 100 ml conical flask. 50 ml of distilled water was added
in 1 ml of K2CrO4 indicator.

5. 0.1N AgNO3 (S ml) was titrated. At the end point, the colour changes from yellow to
brownish red.

6. Blank determination was calculated by using 60 ml distilled water and 1 ml K2CrO4

indicator (B ml).

Calculation

Salt (%) = 250 ml / (10 ml × 25 g) × (S – B) × F × 100

Where,

S = Titration volume of sample (ml)

B = Titration volume of blank (ml)

F = Conversion factor of 1 ml 0.1N AgNO3 to 0.005844 g NaCl

7.2.Annexure II

Principle Secondary Phytochemical Analysis of plant extract, P.amboinicus

The aqueous extract and methanol extract of plant samples were used for the
phytochemical analysis to find the phytochemical constituents in Indian borage.

1. Qualitative analysis

Qualitative analysis is concerned with the presence or absence of phytochemicals.

Test for Phenols (Ferric Chloride test)

1 ml of sample is dissolved in 2 ml of distilled water. To this, a few drops of 2% ferric

chloride solution is added. A dark green color indicates the presence of phenol.

Test for Tannins (Ferric Chloride test)

1 ml of sample is dissolved in 2 ml of distilled water. To this, a few drops of 2% ferric

chloride solution is added. A dark green colour indicates the presence of tannin.

Test for Flavonoids

1 ml of sample is mixed with 10% ammonium hydroxide or ammonium chloride. The
formation of yellow fluorescence indicates the presence of flavonoids.

Test for Saponins

1 ml of sample was mixed with 20 ml of distilled water in a test tube. It was shaken well
for 15 minutes. After 15 minutes, foam is formed for about 2 cm. It indicates the presence
of saponins.

Test for Alkaloids (Wagner’s test)

1 ml of sample and 2 ml of Wagner’s reagent (1 g of Iodine + 2 g of Potassium iodide + 10

ml of distilled water) were added in a test tube. Reddish brown colour appears. It
indicates the presence of alkaloids

2. Quantitative analysis

Quantitative analysis accounts for the quantity or the concentration of the phytochemical
present in the plant sample.

Test for Tannin

It was determined by slightly modifying, Folin & Ciocalteau method. 0.5 ml of sample
and 3.75 ml of distilled water was taken in a test tube. To this, 0.25 ml of folin’s reagent
and 0.5 ml of 35% sodium carbonate solution was added. The absorbance was measured at
725 nm with tannic acid as the standard solution.

Test for Phenol

It was determined by slightly modifying, Folin & Ciocalteau method. 0.2 ml of sample
was taken in a test tube. To this, 0.8 ml of folin’s reagent and 2 ml of 75% sodium
carbonate solution was added. The total content is diluted to 7 volumes with distilled
water and incubated for 2 hours. The absorbance was measured at 765 nm, with gallic acid
as the standard solution.

Test for flavonoids

0.5 ml of sample was taken in a test tube. To this, 3 ml of Ethyl acetate and 3 ml of
Ammonia solution was added. The absorbance was measured at 490 nm.
TEST FOR PRINCIPLE SECONDARY PHYTOCHEMICALS OF P.AMBOINICUS

Qualitative analysis of the plant extract (P.amboinicus) for phenols, tannins,

flavonoids, Saponins and Alkaloids

(1) Phenol (Methanol extract), (3) Tannins (Methanol Extract), (5) Saponins (Methanol extract),
(2) Phenol (Aqueous extract); (4) Tannins (Aqueous extract); (6) Saponins (Aqueous extract);
(7) Alkaloids (Methanol extract), (9) Flavonoids (Methanol extract),
(8) Alkaloids (Aqueous extract); (10) Flavonoids (Aqueous extract).
Quantitative analysis of the plant extract (Plectranthus amboinicus) for phenols,
tannins and flavonoids

(1) Phenol (Methanol extract), (3) Tannins (Methanol Extract), (5) Flavonoids(Methanol extract),
(2) Phenol (aqueous extract); (4) Tannins (Aqueous Extract); (6) Flavonoids (Aqueous extract).