14824 P. Saravana Bhavan et al./ Elixir Bio Tech.

58 (2013) 14824-14828

Available online at www.elixirjournal.org

Bio Technology
Elixir Bio Tech. 58 (2013) 14824-14828

Effects of Piper longum, Piper nigram and Zingiber officinale on survival,

growth, activities of digestive enzymes and contents of total protein, vitamins
and minerals in the freshwater prawn Macrobrachium rosenbergii
P. Saravana Bhavan*, C. Saranya, N. Manickam, T. Muralisankar, S.Radhakrishnan and V. Srinivasan
Department of Zoology, Bharathiar University, Coimbatore – 641046, Tamilnadu, India.

ARTICLE INFO A B ST R A C T
Ar t i cl e h i st o ry : In this study, Piper longum (long pepper), Piper nigram (black pepper) and Zingiber
Received: 6 April 2013; officinale (dried zinger) were incorporated with artificial feeds formulated to understand
Received in revised form: their effects on survival, growth, activities of digestive enzymes and biochemical
7 May 2013; constituents in Macrobrachium rosenbergii post larvae (PL). These herbs were
Accepted: 9 May 2013; independently incorporated with basal ingredients at a concentration of 5% each, and fed to
M. rosenbergii PL (1.56±0.08 cm; 0.074± 0.02 g) for a period of 60 days under laboratory
K e y w or d s condition with triplicates. A significant improvement (P<0.05) in survival and growth
Prawn, performance (weight gain, specific growth rate, food conversion ratio), elevation in activities
Long pepper, of digestive enzymes (protease, amylase and lipase), increased concentrations of total
Black pepper, protein, non-enzymatic antioxidants (vitamins C and E), and mineral salts (sodium and
Dried zinger, potassium) were recorded in P. longum incorporated feed fed PL followed by P. nigram and
Growth, Z. officinale when compared with control. Polypeptide bands of molecular weight between
Survival. 116-14 kDa were resolved in the muscle of PL fed with control as well as herbs incorporated
feeds. Generally, there were eleven Coomassie blue stained protein bands (116, 58, 51, 45,
38, 33, 25, 18, 17, 16 and 14kDa) were calculated in herbal incorporated test samples. The
general comparison between control and experimental groups revealed that there was more
number of bands resolved in herbals incorporated feeds fed PL. Therefore, it is suggested
that these medicinal herbs have their own influence in protein synthesis. In this study, the
overall results indicated the fact that these herbs have acted as appetizers and hence,
enhanced the secretion of digestive enzymes, which facilitated efficient digestion, absorption
of nutrients and favoured for general health, which in turn ultimately produced better
survival and growth of M. rosenbergii PL. Therefore, these herbs can be taken as feed
additives in sustainable development of freshwater prawn culture.
© 2013 Elixir All rights reserved.

Introduction the control showed only 25% survival (Direkbusarakom et al.

The giant river prawn, Macrobrachium rosenbergii is 1996). Herbs such as, Solanum trilobatum, Psoralea corylifolia
dominating in India, and it is one of the major contributors of and Clematis paniculata enriched Artemia fed Penaeus
national economy. It is good source for protein, essential amino monodon had reduced vibrio species (Citrasu et al. 2002, 2009).
acids and polyunsaturated fatty acids, it is very low in fat, and Many plants have antibacterial and antifungal properties. For
therefore, it can be used as a healthy choice of food for human instance, the extract of Ocimum basilicum has controlled
(Bhavan et al. 2010). The successful prawn culture depends on infection of Aspergillus flavus and Fusarium oxyspoum
quality live feeds and nutrient rich commercial feeds. Both are (Adiguzel et al., 2005). Herbal compounds have the ability to
very costly and non-affordable to small farmers. Therefore, low inhibit the generation of oxygen anions and scavenge free
cost artificial feeds are much needed for promoting inland radicals, hence reduces the effects due to stress.
aquaculture of freshwater prawns. Piper longum (long pepper) is a component of medicines
Nowadays, herbals are widely used in veterinary and human which is reported as good remedy for treating gonorrhea,
medicine. The herbal active principles in the diets induce the menstrual pain, tuberculosis, sleeping problems, respiratory tract
secretion of the digestive enzymes. It would stimulate the infections, chronic gut-related pain and arthritic conditions. It
appetite and ultimately increased the food consumption and has antioxidant, antimicrobial, antipyretic, antiallergic,
efficiencies (Bhavan et al. 2011; Shanthi et al. 2012; immunomodulatory and antitumour activities (Joy et al. 2010;
Radhakrishnan et al. 2013). Herbal active principles induce Khushbu, 2011). Other beneficial effects of P. longum include
transcription, which lead to high protein synthesis (Citrasu et al. analgesic and diuretic effects, relaxation of muscles tension and
2002, 2009). Several plant products found to have potent alleviation of anxiety. Alkaloids, piperine, piper longumine and
antiviral activity against fish and shrimp viruses. For example, pipernonaline are the active principles of P. longum.
shrimp fed with ethanolic extract of Clinacanthus nutans Piper nigrum (black pepper) has many medicinal uses,
showed 95% survival rate against yellow head virus (YHV), but including the ability to control worm infestations, and to provide

Tele:
E-mail addresses: [email protected]
© 2013 Elixir All rights reserved
14825 P. Saravana Bhavan et al./ Elixir Bio Tech. 58 (2013) 14824-14828

relief for a number of ailments including asthma, cough, heart groundnut oil cake (25g), and wheat bran (10g). Tapioca flour
diseases, throat inflammations, night blindness, urinary (7g) and egg albumin (5 ml/ 100 g) was added as binding agents.
disorders, tooth and muscle aches, inflammations, snake bites, Before addition of the egg albumin the mix was cooked for 15
eye diseases, cholera, and swoons. It has antibacterial, minutes at 95-100oC and cooled at room temperature. Sunflower
antifungal, antioxidant, antihepatotoxic, antimalarial and oil (2 ml) was added as lipid source. Vitamin B-complex with
antileukemic activities (Pundir et al. 2010; Bai et al. 2011). vitamin-C (1 %) was also mixed. A pinch of salt was also added.
The roots of pepper also have medicinal qualities, as a stomach The basal diet has 3271 k.cal/kg of energy. With this, each
anesthetic, analgesic, muscle relaxant, digestive stimulant, herbal powder, P. longum, P. nigram and Z. officinale was
antiseptic, diuretic, sudorific (diaphoretic, promotion of separately added at a concentration of 5% each and mixed well.
sweating), anxiolytic, and as a hypnotic (Nelson & Eger, 2010). The dough was manually pressed through a locally
Externally it is valued for its rubifacient properties and as a local manufactured feed pelletizer. The pellets were dried in well-
application for relaxed sore throat, piles and some skin diseases aerated place under shade for 2 days until they became
(Pullaiah, 2002). In aquaculture, P. nigrum plant extracts posses sufficiently dried. Finally, the pellets were stored in air tight
antibacterial activity against Edwardisellosis agents and bacteria containers and kept in a cool, dry place and fed to M.
tested in ornamental sea anemone, Radisanthus ritteri (Wei et al. rosenbergii PL.
2007). Piperine and piperoyl-piperidine are two major herbal The experiment was conducted in triplicate with 25 PL each
principles. Piper extracts and piperine possess inhibitory (PL30-90; 1.56±0.18 cm; 0.074± 0.02 g) in 30 plastic aquaria of
activities on prostaglandin (Mishra, 2010). It is also used as a 25 L capacity (each herb represents three diets with a common
purgative, an antidote to snake-bite and scorpion-sting (Sumy et control). Each group was fed with a specific herb incorporated
al. 2000). diet ad libitum (1g per aquarium) for a period of 60 days. The
Zingiber officinale (dried ginger) is used as ayurvedic water medium was renewed daily by siphoning method without
medicine to cure indigestion, stomachache, abdominal pain, severe disturbance to the PL and aerated. The faecal matter,
disorders of gallbladder, hyperacidity, hypercholesterolemia, unfed feed, and exuvia if any were collected separately. Similar
hyperglycemia, morning sickness, prevention of motion experimental set-ups were maintained few times as and when
sickness, and nausea (Yohannes, 2010). It possesses required to study various parameters. The nutritional indices
antimicrobial, antipyretic, ectoparasitic, antioxidant, anticancer were calculated from the initial and final morphometric data.
and immunomodulatory activities (Yusof et al. 2002; Sasidharan Weight Gain (WG) = Final weight (g) – Initial weight (g)
et al. 2010; Famurewa et al. 2011). It also used as an antiemetic Total quantity of feed consumed (dry wt.)
agent (Phillips et al. 1993). In aquaculture, the rainbow trout Feed Conversion Ratio (FCR) = --------------------------------------
(Oncorhynchus mykiss) fed with 1% ginger extract exhibited Live weight gain (g)
increased phagocytic activity of blood leukocytes (Chung & No. of live prawns
Secombes, 1987). Its active principles are gingerol, zingerone Survival Rate (SR) = ------------------------------- X 100
and shogaol. No. of prawns introduced
The present study was aimed to understand the influence of Activities of digestive enzymes, such as protease, amylase
incorporation of medicinal herbs, P. longum, P. nigrum and Z. and lipase were assayed on final day of feeding trial. The
officinale at a concentration of 5% each with basal ingredients whole flesh except eye stalk, appendages and exoskeleton was
on the survival rate growth performance, activities of digestive homogenized in ice cold distilled water and centrifuged at
enzymes, and the basic biochemical constituents, such as total 10,000 rpm under 40C for 20 minutes. The supernatant was used
protein, vitamins (C and E), minerals (sodium, Na+ and as crud enzyme source. Protease activity was estimated by the
Potassium, K+) and protein profiles in M. rosenbergii PL. method of Furne et al. (2005). One unit of enzyme activity
Materials and Methods represents the amount of enzyme required to liberate one µg of
The post larvae (PL-15) of the freshwater prawn, M. tyrosine per minute under assay conditions. Amylase activity
rosenbergii were procured from Aqua Hatcheries, Happy bay was assayed followed by the method of Bernfeld (1955) in
annexe, Mugaiyur Village, ECR, Cheyyur Taluk, Kanchipuram which the increase in reducing power of buffered starch solution
District, Tamilnadu, India. They were transported to the was measured. The specific activity of amylase was calculated
laboratory in polythene bags filled with hatchery water (pH, as mg of maltose liberated/ g of protein/ h (mg/g/h). Lipase
6.8; total dissolved solids, 1.2 g/L; dissolved oxygen, 6.5 mg/L; activity was assayed by the method of Furne et al. (2005). The
BOD, 42.0 mg/L; COD, 140.0 mg/L; ammonia, 1.20 mg/L) and amount of free fatty acid released per unit time was estimated by
acclimatized for two weeks (up to PL-30) using ground water the amount of NaOH required to maintain pH constant and
(pH, 7; total dissolved solids, 1200 mg/L; dissolved oxygen, 7.2 represented as mille equivalents of alkali consumed.
mg/L; BOD, 30.0 mg/L; COD, 125.0 mg/L; ammonia, 0.028 The concentration of total protein in the muscle was
mg/L). During acclimatization, the PLs were fed with boiled egg determined by the method of Lowry et al. (1951) using alcohol
albumin and Artemia nauplii alternatively twice a day, and latter precipitated sample. For estimation of soluble protein, the
they were only maintained with commercially available scampi muscle tissue samples were first defrosted in phosphate buffer,
feed. Water was adequately renewed daily. At the same time, the pH, 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2
faecal matter and unfed feed were removed. The medium was mM KH2PO4), homogenized under ice cooled condition and
adequately aerated. centrifuged at 1500 rpm at 4°C for 5 min. The soluble protein
Ten different types of iso-caloric diets containing content in supernatant was determined by following the method
approximately 40% protein, 33% carbohydrate and 12% lipid of Lowry et al. (1951). SDS-PAGE was performed on vertical
were formulated by using the following basal ingredients chosen slab gel with 4% stacking and 10% separating gels (Laemlli,
on the basis of their nutritional status and year round availability 1970). Samples of control and the best concentration of each
in the local market, such as fishmeal (25%), soy meal (25g), herb incorporated feed fed PL were taken into consideration.
14826 P. Saravana Bhavan et al./ Elixir Bio Tech. 58 (2013) 14824-14828

Protein marker consisted of six different molecular weights arvensis incorporated feeds (Bhavan et al. 2011, 2012; Shanthi
(Medox-Bio Pvt. Ltd., India), such as β-galactosidase (116 kDa), et al. 2012).
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic Protein Profile
anhydrase (29 kDa), soya bean trypsin inhibitor (20 kDa) and Polypeptide bands of molecular weight between 116-14
lysozyme (14 kDa) was also run to calculate the kDa of various kDa were resolved in the muscle tissue of PL fed with P.
bands resolved in unknown sample. The banding pattern longum, P. nigrum and Z. officinale incorporated feeds, and
between control and test was compared by information on control as well (Fig.1). Generally, there were eleven Coomassie
apparent molecular masses and intensity. blue stained protein bands (116, 58, 51, 45, 38, 33, 25, 18, 17,
Concentrations of vitamin-C (ascorbic acid) and vitamin-E 16 and 14kDa) were calculated in herbal incorporated test
(α-tocopherol) were estimated in the muscle of PL following the samples against the standard markers of 116, 66, 45, 29, 20 and
method of Roe & Kuether (1943) and Baker et al. (1980) 14 kDa, which represent β-galactosidase, Bovine serum
respectively. Contents of minerals, Na+ and K+ were estimated in albumin, ovalbumin, carbonic anhydrase, soyabean trypsin
the muscle of PL following the simple flame photometric inhibitor and lysozyme respectively. In P. longum incorporated
method of Jeffery et al. (1989) by using Elico CL 220 flame feed fed PL all these bands were stained more intensely than P.
photometer. The values are calculated by adopting the following nigram, and Z. officinale incorporated feeds fed PL. Major
formula. changes in experimental groups were seen in the region between
Sample reading Standard concentration 33kDa-25kDa, and between 17kDa-14kDa. In the case of
Na+/ K+ Content (mg) = -------------------- x --------------------------- x Purity of NaCl/ K
Standard reading Sample concentration control, the 116kDa, 25kDa, 18kDa and 17kDa were completely
The data were subjected to statistical analyses by adopting lack of stained bands. The general comparison between control
‘two-tailed paired sample t-test’ through SPSS software (version and experimental groups revealed that there was more number of
16) and the significance was noted. bands resolved in herbals incorporated feeds fed PL. Therefore,
Results and Discussion it is suggested that these medicinal herbs have their own
Morphometry and nutritional indices influence in protein synthesis.
The length, weight, weight gain, survival rate and specific
growth (SGR) rate were found to be significantly increased
(P<0.05) in experimental feeds fed PL groups when compared
with control. The reverse trend was seen in feed conversion ratio
(FCR), which indicates the quality of the experimental feeds.
Among the three herbs used, P. longum incorporated feed fed
PL showed the best performance followed by P. nigrum and Z.
officinale (Table 1). The increase in growth performance has
also previously been reported in M. rosenbergii fed with
Ocimium sanctum, and Withania somnifera incorporated feeds
(Bhavan et al. 2011), Andrographis paniculata, Cissus
quadrangularis, and Eclipta alba incorporated feeds (Shanthi et
al. 2012) and greens, Murraya koenigii, Coriandrum sativum
and Menthe arvensis incorporated feeds (Bhavan et al. 2012).
Digestive enzymes M, marker proteins; C, control; 1, P. longum ; 2, P. nigrum; 3,
The activities of digestive enzymes, such as protease, Z. officinale
amylase and lipase were found to be significantly increased Figure 1: Protein profile of the muscle tissue of M.
(P<0.05) in experimental feeds fed PL groups when compared rosenbergii fed with P. longum, P. nigrum and Z. officinale
with control. Among the three herbs used, P. longum incorporated feeds
incorporated feed fed PL showed the best performance followed The herbal principles may enhance protein synthesis as
by P. nigrum and Z. officinale (Table 1). Increase in activities of reported in L. rohita (Johnson & Banerji, 2007) fed with S.
protease, amylase and lipase has also previously been reported portulacastrum supplemented diet. The herbal growth promoters
in M. rosenbergii PL fed with A. paniculata, C. quadrangularis, may induce transcription and enhanced protein synthesis
E. alba, M. koenigii, C. sativum and M. arvensis incorporated (Citarasu, 2009; Poonkodi et al. 2012).
feeds (Shanthi et al. 2012; Bhavan et al. 2012). The increased Concentrations of vitamins and minerals
activities of digestive enzymes reflect the fact that the appetite Concentrations of non-enzymatic antioxidants, vitamin-C
was induced, which in turn influenced food consumption, and and vitamin-E, and minerals, Na+ and K+ were found to be
facilitated effective digestion, absorption and ultimately growth significantly increased (P<0.05) in experimental feeds fed PL
of PL. groups when compared with control (Table 1). Among the three
Total protein herbs used, P. longum incorporated feed fed PL showed the best
Concentration of total protein was found to be significantly performance followed by P. nigrum and Z. officinale (Table 1).
increased (P<0.05) in experimental feeds fed PL groups when Elevation in concentrations of vitamin-C and vitamin-E has
compared with control. Among the three herbs used, P. longum previously been reported in M. rosenbergii PL fed with A.
incorporated feed fed PL showed the best performance followed paniculata, C. quadrangularis, E. alba, M. koenigii, C. sativum
by P. nigrum and Z. officinale (Table 1). Increase in total and M. arvensis incorporated feeds (Shanthi et al. 2012; Bhavan
protein has also previously been reported in M. rosenbergii PL et al. 2012).
fed with O. sanctum, W. somnifera, A. paniculata, C.
quadrangularis, E. alba, M. koenigii, C. sativum and M.
14827 P. Saravana Bhavan et al./ Elixir Bio Tech. 58 (2013) 14824-14828

Table 1: Morphometric data, nutritional indices, biochemical constituents, activities of digestive enzymes,
concentration of vitamins and minerals in M. rosenbergii PL fed with medicinal herbs (Z. officinale, P. nigrum
and P. longum) incorporated feeds
Control Experiment
Parameters
P. longum P. nigrum Z. officinale
Morphometric data Length (cm) 2.20±0.10 4.36±0.28 3.84±0.01 3.27 ±0.06
Weight (g) 0.20±0.01 0.66±0.02 0.52±0.01 0.39±0.01
Weight gain (g) 0.131±0.09 0.592±0.09 0.446±0.07 0.316±0.08
Nutritional indices Survival rate (%) 73.33±0.65 96.66±0.14 90.00±0.8 83.33±1.00
Specific growth rate 0.211±0.10 0.723±0.09 0.616±0.08 0.491±0.05
Feed conversion ratio 4.8±0.96 1.92±0.23 2.4±0.67 3.03±0.89
Protease 1.33±0.32 4.83±0.25 4.38±0.04 4.37±0.01
Digestive enzymes
Amylase 1.72±0.27 5.32±0.65 3.50±0.50 3.46±0.65
(U/ mg protein)
Lipase 0.15±0.07 0.70±0.10 0.68±0.01 0.67±0.17
Total protein (mg/g wet. wt) 91.02±193 423.66±0.04 337.6±4.00 344.97±1.97
Vitamins Vitamin C 24.00±1.00 58.70±2.28 50.01±2.10 48.49±0.50
(µmol/mg protein) Vitamin E 26.50±0.50 46.38±1.49 39.83±0.17 37.04±1.04
Minerals Na+ 5.44±0.98 19.52±1.38 17.17±0.98 16.26±0.88
(mg/g) K+ 4.07±1.01 11.74±0.80 8.62±1.90 7.67±056
Initial length: 1.56±0.08 cm; Initial weight: 0.074±0.02 g.
Length and weight were randomly measured from five individuals and the mean value was considered as a single observation and
three such observations were made.
Each value is mean ± SD of three individual observations.
All the values are significant at P<0.05

Increase in concentrations of Na+ and K+ has also previously Baker H, Frankel O (1980). Plasma-α-tocopherol in man at
been reported in M. rosenbergii PL fed with Alteranthera various time intervals after ingesting free or acetylated
sessilis, E. alba and C. quadrangularis incorporated feeds tocopherol. Nutrition Reports International 21, 531-536.
(Radhakrishnan et al. 2013). Bendich A, Machlin LJ, Scansurra O, Byrton GW, Wayner
Vitamin C and E (ascorbic acid and α-tocopherol DDM (1986). The antioxidant role of vitamin C. Advances in
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Trabor, 1990). (2010). Basic biochemical constituents and profiles of amino
The macro elements sodium and potassium in animal are acids in the post larvae of Macrobrachium rosenbergii fed with
connected with the regulation of intracellular osmotic pressure spirulina and yeast enriched Artemia. Journal of Scientific
and the maintenance of acid-base balance. Particularly, Research 2, 539-549.
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It is evident that P. longum, P. nigrum and Z. officinale rosenbergiipost larvae fed with Ocimum sanctum (Tulsi) and
have the ability to induce secretion of protease, amylase and Withania somnifera (Ashwagandha) incorporated feeds.
lipase in M. rosenbergii PL, which in turn associated with active International Journal of Biological Research and Development
principles of these herbs and minerals suggested enhanced 1, 34-53.
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suggested effective scavenging of free radicals, which ultimately herbal greens, Murraya koenigii, Coriandrum sativum and
resulted in better survival and growth of M. rosenbergii PL.. Menthe arvensis on growth performance of the freshwater prawn
Therefore, these herbs can be utilized for development of Macrobrachium rosenbergii post larvae. Research Journal of
sustainable culture of Macrobrachium. Biotechnology 7, 149-157.
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