An efficient protocol for mass multiplication of Centella asiatica (L.) Urban and determination of its phenolic content

Plants are natural source of medicine and used in curing diseases. The medicinal plants have been used as the herbal remedies. At present use of natural products in treating diseases are increasing. Herbal drugs have achieved much popularity due to increasing awareness towards personal health which leads to maintained health through the natural products [1]. The high public demands of these medicinal plants required its conservationthrough the micropropagation as well as enhancement of secondary metabolites through the callus culture, suspension cultures and treatment of elicitor’s molecule [2]. Centella asiaticawhich is also known as Indian pennywort are small herbaceous annual plant widely distributed in India, Malaysia, SriLanka, Indonesia, and other parts of Asia [3,4]. The plant belongs to the family Apiaceae [5]. The extract of Centella asiaticais used for wound healing [6] as well as various skin diseases viz: leprosy, lupus, varicose ulcer,eczema, psoriasis and female genital urinary tract infections [7]. Ethanolic extract of Centella has been well elucidated for antibacterial activity [10]. Apart from antibacterial activity, essential oil of C. asiatica is endowed with antimicrobial activity [9,11].Crude methanolic extract as well as hydroalchoholic activity showed antioxidant properties [8]. Centella asiaticais assumed to be effective on the connective tissues by strengthened weakened viens[12]. Triterpene of C.asiatica also showed antidepressant activity [13].Centella asiatica of Asiatic region enhances GABA in cerebral cortex which depicts its conventional anxiolytic and anticonvusalant property [7,14]. The major constituents of these medicinal plants are madecassid acid, asiatic acid, centellasaponins as well as three types of asiaticoside viz: asiaticoside, asiaticoside A and asiaticoside B [15]. Because of great demands of these bioactive molecules, researchers focused to develop plant tissue culture techniques to increase the number of plantlets as well as to enhance the secondary metabolite production through the callus culture [16, 28]. Therefore, the major thrust of present investigation was to standardize a protocol for micropropagation of this valuable medicinal plant in B5 media. We also determined bioactive molecules productioninvivo and invitrocondition.


INTRODUCTION
Plants are natural source of medicine and used in curing diseases. The medicinal plants have been used as the herbal remedies. At present use of natural products in treating diseases are increasing. Herbal drugs have achieved much popularity due to increasing awareness towards personal health which leads to maintained health through the natural products [1]. The high public demands of these medicinal plants required its conservationthrough the micropropagation as well as enhancement of secondary metabolites through the callus culture, suspension cultures and treatment of elicitor's molecule [2]. Centella asiaticawhich is also known as Indian pennywort are small herbaceous annual plant widely distributed in India, Malaysia, SriLanka, Indonesia, and other parts of Asia [3,4]. The plant belongs to the family Apiaceae [5]. The extract of Centella asiaticais used for wound healing [6] as well as various skin diseases viz: leprosy, lupus, varicose ulcer,eczema, psoriasis and female genital urinary tract infections [7]. Ethanolic extract of Centella has been well elucidated for antibacterial activity [10]. Apart from antibacterial activity, essential oil of C. asiatica is endowed with antimicrobial activity [9,11].Crude methanolic extract as well as hydroalchoholic activity showed antioxidant properties [8]. Centella asiaticais assumed to be effective on the connective tissues by strengthened weakened viens [12]. Triterpene of C.asiatica also showed antidepressant activity [13].Centella asiatica of Asiatic region enhances GABA in cerebral cortex which depicts its conventional anxiolytic and anticonvusalant property [7,14]. The major constituents of these medicinal plants are madecassid acid, asiatic acid, centellasaponins as well as three types of asiaticoside viz: asiaticoside, asiaticoside A and asiaticoside B [15]. Because of great demands of these bioactive molecules, researchers focused to develop plant tissue culture techniques to increase the number of plantlets as well as to enhance the secondary metabolite production through the callus culture [16,28]. Therefore, the major thrust of present investigation was to standardize a protocol for micropropagation of this valuable medicinal plant in B5 media. We also determined bioactive molecules productioninvivo and invitrocondition.

Source of Explant and Surface Sterilization
Germplasmsof Centella asiatica were collected from different ecological niches of Bihar such as Betiah, Purnia, Motihari and Patna, planted in garden of PG Department of Botany, Patna University Patna. One year grown plants were selected as explants for micropropagation on the basis of their morphological features and vigorous growth. The germplasm of Motihari showed maximum growth in leaf as well as size of leaf and stem as compared to other districts of Bihar. Therefore, thegermplasm of Motihari District of Bihar for the Micropropagation of Centella asiatica. Stem and leaf were cut from the mother plantand washed under the running tap water for 30 minutes. Afterthen washed explants were soaked in 5% (v/v) teepol for 8 min and washed properly with double distilled water, followed by surface sterilization by 0.1% (w/v) freshly prepared mercuric chloride for 5 minute and repeated washing 3-4 times with sterile double distilled water under laminar airhood. Explants were cut in 2-2.5 cm in small pieces.

Inoculation of Explants in B5 Media
The surface sterilized explants were dried out on autoclaved filter paper and further inoculated in plant tissue culture tubes (150×25 mm) under laminar air hood containing20-25 ml of B5 media [17] augmented with different combination and composition of auxins such as 2, 4-D, NAA and cytokinins BAP and kinetin. pH of media was adjusted with the help of NaOH and HCl prior to autoclave at 1.05 kg/cm 2 pressure and 121 0 C temperature for15 min. The uniform culture condition was maintained in plant tissue culture laboratory of department at temperature 25 ± 2 0 C under a photoperiod of 16/8 hr with relative humidity (RH) 60-70 % and light intensity of 35-40 µmolm -2 S -1 (white fluroscent light). Twenty replicates raised per treatment were repeated three times. Values represent means ± SE of 10 explants per culture vessels. The cultures were maintained by subculturing at regular interval of 4 weeks. Data were collected after inoculation of 4 week. Randomly selected in vitro grown calluses were further transferred in freshly prepared shooting B5 media supplemented with BAP and NAA for proliferation of callus into shoots. The number of shoots per callus and length of shoots were recorded after 4 week. When the shoots achieved the length of 2-2.5 cm, they were further transferred in rooting media fortified with BAP and NAA in tissue culture jar (PW1300, Himedia). Data were collected after 4 week of inoculation in shooting and rooting media. Percentage of shooting, number of roots and length of root (in cm) were also recorded.Moisture contents of randomly selected calluses were determined by the drying it in incubator at 30 0 C. Firstly we measured the weight of fresh calluses then kept the material in Petri plates and placed it in incubator for 72 hours. The moisture of calluses wasdetermined using the formula, % of moisture content = (FC-P) -(FD-P) ×100/ (FC-P), where, P is weight of empty Petri plates, FC is weight of Petri plates with fresh calluses and FD is weight of Petri plates with dry calluses.
The total phenolic content of Invitro grown callus and In vivo explants was studied by the method of Govarthanan etal. with slightly modification [4]. The folin-ciocalteu's (FC) reagent was used fordetermination of total phenolic. One ml of methanolic extract and nine ml of double distilled water was added in 25 ml of conical flask. One ml of folin -ciocalteu reagent was added in the mixture with constant stirring. After 5 min 10 ml of 7 % (Na 2 CO 3 ) was added in the mixture and volume was made up to 25 ml with sterile distilled water. The reaction was incubated for 90 min on room temperature and absorbance was determined at 550 nm with the help of an ultraviolet (UV) visible spectrophotometer. Total phenolics content was expressed as mg gallic acid equivalents (GAE)/gm of C. asiatica extract. Meanwhile after induction of roots plantlets were removed from the media and cleaned with double distilled luke warm water for removal of agar and transferred into sterile plastic pots containing sand, soil, vermiculatie (1:1:1) covered with sterile polythene bags. In order to acclimatize plants into field, after 2 week polythene bags were opened and transferred in earthen pots filled with 3:1 mixture of soil and vermicompost. Invitro grown plantlets were maintained in green house in normal day-night condition.

Statistical Analysis
A complete random block designed was design for all investigations such as callus culture, shoot induction, rooting and plant regeneration. Data were calculated on the basis of number of explants used. Twent replicates per treatment were repeated three times and values represented by mean±SE ten explants per culture vessels.

RESULTS AND DISCUSSION
The stem and leaf explants were inoculated in B5 media augmented with different combination and composition of BAP, kn NAA and 2, 4-D. The results have been presented in (Table 1) and Figure 1).After one week of inoculation callusesturned up reddish due to secretions of secondarymetabolites.It was observed that B5 media augmented with BAP concentration ranges from 0.1 to 2.5 mg/l and NAA ranges 0.1-0.5 mg/l lead to formation of callus from leaf and stem explants ( Figure 1). The calli obtained from the leaf were green, brown, compact and friable in nature.The same type of calli was also obtained from stem explants in B5 media fortified with BAP 0.1-2.5 mg/l and NAA 0.1-0.5 mg/l.Higher concentration of plant growth regulators leads to formation of brown and friable calli (Tables1 and Figure 1 (Table 1) while maximum duration was seventeen days for induction (Table 1) of callus responses in the stem explants. BAP 2.5 mg/l augmented with NAA 0.5 mg/l showed 10 out of 10 culture vessels in both explants, whereas kn 2 mg/l supplemented with 0.2 mg/l 2, 4-D results 8 out of ten culture vessels in stem and leaf explants. BAP showed 100% calluses response and Kn showed 80% response.The moisture content of calluses obtained from both the explants was determined by drying the calluses in incubator ( Table 2). The leaf explants showed minimum moisture content 59% and maximum 91% while in stem minimum moisture content

Multiplication of Shoots
Calluses derived from stem and leaf explants were further inoculated in shooting media.BAP (0.5-1.5 mg/l) fortified with NAA (0.1-0.5 mg/l) in B5 media leads to formation of shoots in Centella asiatica. BAP 1.5 mg/l and NAA 0.5 mg/l has maximum shoot length 2.01cm whereas BAP 01 mg/l and NAA 0.2 mg/l have minimum shoot length of 0.49 cm. Number of shoots per explants was minimum at BAP (01 mg/l) and NAA(0.2 mg/l) while maximum at BAP (1.5 mg/l) and NAA (0.5 mg/l) (Table3 Figure 2).It has been observed that low concentration of plant growth regulators showed poor growth and high concentration showed green shoots with branching. After transfer of callus in shooting media shoots were regenerated after four week of inoculation.

Root Induction
After shooting, 1.5 -2 cm long shoots were transferred in full strength B5media augmented with BAP 0.1 mg/l and NAA 0.1-0.5 forroot induction. The results have been presented in (Table 4 and Figure 3). It has been shown that 0.1 mg/l BAP fortified with NAA 0.5 mg/l showed 100 % rooting, highest root per shoot 11.6 cm and mean root length 1.86 cm. However percentage of rooting, root length and number of roots per shoot varied among the different media composition. It has been observed that increasingthe concentration of NAA enhances the percentage rooting, number of roots per shoot as well as root length. No resultsof rooting were observed in BAP augmented with IBA.

Acclimatization of Plantlets
For acclimatization well developed plantlets were isolated from rooting media and washed with luke warm double distilled water for removal of agar and subjected to hardening. Plantlets were transferred in soil mixture containing vermiculatie sand and soil (1:1:1) in plant growth chambers for 2 week (Figure 4). The plantlets were transferred into earthen pots into green house, potted in 3; 1 vermicompost and soil for another 2 week. After then plants were kept outside under full sunlight which is resulted into 90% survival.

Analysis of Phenol
Subsequent analysis of total phenolic content was determined In vivo leaf, stem and invitrogrown 40 days old leaf and stem calluses. The percentage yield of total ethanolic alkaloid contents were 11.8%, 12% and 21% in powder tissues of stem, leaf and invitro raised calluses respectively. Calluses contain more alkaloid contents as compared to the explants.The phenoliccontent of calluses was more as compared to leaf and stem explants. The callus contained 0.67 mg/gm dw of phenolic while leaf 0.63 mg/gmdw and stem 0.59 mg/gmdw respectively.

DISCUSSION
Micropropagation protocol represents minimum coast techniques, by which we can obtained high quantity of plants. In micropropagation by using phytohormones reduced expenses and enhances fast and effective plant production. The present investigation standardized a protocol for callus induction and regeneration from stem and leaf explants in Centella asiatica. This experiment determined a protocol for shoot proliferation and root emergence from the callus. The combination of BAP and NAA was essential for callusinduction as well as plant regeneration [2,[18][19][20][21][22][23][24][25][26]. The effectiveness BAP and NAA showed by various researchers in Centella asiatica [30]. BAP and Kn showed significant role in callus induction [21].Combination of Kn with 2, 4-D showed good results which is same as previous findings [27,28]. But the characteristics feature of callus was different. The culture condition, age and type of explants may attribute for this differences.The combination of cytokinin and auxin are very important for callus induction and multiplication of shoots from the explants.
No regeneration was observed without the growth regulators [18]. Similar regeneration was reported in combination of BAP and NAA in B5 media [15,24].Phenols are the important constituents of the medicinal plants possess hydroxyl groups which are mainly responsible for antioxidant property in any plants. Various researches have been conducted on polyphenols and stated that polyphenols showed inhibitory effect against cancer and mutation. The callus contains more amounts of phenolics as compared to explants and leaf has also high amount of phenolic as compared to stems. Callus cultures have ability to attain significant amounts of phenolics compound as compared to the counter parts of In vivo grown plants. Callus grow in nutrient rich media, which is exposed to more carbon Means were represented as means ± SE (10 explants per culture vessels repeated three times) influx than the In vivo grown, it might attribute to influence metabolic flux for the synthesis of increased level of phenolics. Phenolics compounds have redox properties which are also responsible for antioxidant properties. Among the three plant parts (leaf, stem, and callus) of Centella asiatica, the callus showed maximum amounts of total phenolics compound. Plant tissue culture techniques are very important tools for secondary metabolites production. Callus culture has been mostly used for secondary metabolites production against the intact plants. Several researchers have been reported secondary metabolite production through the in vitro culture in various medicinal plants [31][32][33].

CONCLUSION
The callus induction and regeneration of plantlets were easily maintained in B5 media fortified with auxin Viz: NAA, 2, 4-D and cytokinin BAP and NAA. The experiment revealed that combination and composition of plant growth regulators are responsible for callus induction as well as regeneration of plantlets. Combination of BAP and NAA are the best for callus induction over the combination of Kn and 2, 4-D. For callus induction stem is better explants as compared to leaf explants, however leaf contains high amount of phenolics relative to stem. Callusesshowed more amounts of phenolicsas compared to both the explants. Higher of plant growth regulators enhances callus induction as well as shoot multiplication and root induction. B5 medium augmented with BAP and NAA are responsible for shooting and rooting. Rooted plantlets were transferred into field where they achieved 90% establishment successfully. The investigation may be concluded that a simple and efficient protocol was developed for rapid callus induction and regeneration in C. asiatica. The determination of phenolics suggested that the callus possessed high amounts of metabolites as compared to explants. This micropropagatedprotocol might be helpful in conservation of C. asiatica which is under threatened species due to overexploitation.