|Year : 2013 | Volume
| Issue : 1 | Page : 60-65
In vitro pharmacological investigations of Biophytum sensitivum callus extract: Lack of potent activities
Sirigiri Chandrakala1, Kokkanti Mallikarjuna1, Challa Siva Reddy2
1 Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, India
2 Department of Pharmacology, KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, India
|Date of Web Publication||21-Aug-2013|
Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Biophytum sensitivum is an important medicinal plant extensively used in traditional oriental herbal medicines. Though medicinal use of this plant is known, the active principles responsible such property is not known. Pharmacological screening of this plant may lead to discovery of new activity with new mode of treatment. Hence, screening for in-vitro pharmacological activities of methanolic callus extracts of Biophytum sensitivum has been carried out. Experiments were designed according to the standard methods and processes. Materials and Methods: Leaf cutting derived callus on MS medium supplemented with BA (Benzyl adenine) 1.0 mg/l + NAA (1-naphthaleneacetic acid) 1.0 mg/l is used as a source, and compounds were extracted from dried callus using methanol solvent with Soxhlet apparatus. Results: The callus extract has shown antioxidant activity, in-vitro inhibition of enzyme activities like α-glycosidase, acetyl cholesterase, and tyrosinase, but potency was found to be low. The Graph pad Prism Version-5 software is used to analyze data in the form of Figures. Conclusion: For the first time, we are reporting in-vitro pharmacological screening of methanolic callus extracts of Biophytum sensitivum.
Keywords: Acetyl cholinesterase, anti-oxidant, Biophytum sensitivum callus extracts, Ttyrosinase, α-glucosidase
|How to cite this article:|
Chandrakala S, Mallikarjuna K, Reddy CS. In vitro pharmacological investigations of Biophytum sensitivum callus extract: Lack of potent activities. J Pharm Negative Results 2013;4:60-5
|How to cite this URL:|
Chandrakala S, Mallikarjuna K, Reddy CS. In vitro pharmacological investigations of Biophytum sensitivum callus extract: Lack of potent activities. J Pharm Negative Results [serial online] 2013 [cited 2020 Aug 13];4:60-5. Available from: http://www.pnrjournal.com/text.asp?2013/4/1/60/116760
| Introduction|| |
Biophytum sensitivum (Linn.) DC. (Syn.Oxalis sensitiva Linn.) is a small annual herb belonging to the family Oxalidaceae. It grows in shady places in dry parts of India during the rainy season. It is extensively used in traditional oriental herbal medicines.  Biophytum sensitivum flower is used in athapoo, a special floral formation, that adores courtyards and public places during Onam festival in Kerala. Since the plant is reputed for its folkloric uses in various diseases,  it draws our attention for it's in-vitro pharmacological screening.
Plant tissue culture technology has resulted in the production of many pharmaceutical substances for new therapeutics. Successful attempts to produce some of these valuable pharmaceuticals in relatively large quantities by cell cultures are illustrated. The pharmacological drugs are either too expensive or have undesirable side effects contraindications.  Pharmacologically, the Biophytum plant has been investigated for its hypoglycemic,  anti-inflammatory,  hypocholesterolemic,  and anti-cancer effect.  It is a known traditional remedy for the treatment of diabetes  and anti-tumor activities.  It possess a wide spectrum of medicinal properties for asthma, snakebites, stomachalgia and phthisis, positive effects in inflammatory diseases and antioxidant activity,  and leaf extracts with anti-bacterial activity.  Biophytum sensitivum extracts lowered blood sugar on streptozotocin and nicotinamide-induced diabetes in rats. 
Though the mature wild plant has been screened for various pharmacological activities, in-vitro screening for various pharmacological activities using callus is lacking. As the in-vitro callus represent good source of secondary metabolites, screening of such callus may give insight into the presence of unknown compounds with new activity.  Hence, the present study was undertaken to scientiﬁcally investigate various in-vitro pharmacological activities like antioxidant and inhibition assays of α-glucosidase, acetyl cholinesterase and tyrosinase of methanolic callus extracts of Biophytum sensitivum. Our study revealed that, indeed it shows antioxidant and inhibition activities of various enzymes indicating that it can be explored as antioxidant chemicals.
| Materials and Methods|| |
Preparation of plant material
The fresh matured plants (100 no.) of the Biophytum sensitivum collected from A.N.U Campus, Guntur District were used as a source of explants. The leaf explants were excised into 1 cm long segments and were washed with liquid detergent (5% Teepol, Qualigens, India) followed by Bavistin (1% w/v) for 3 min, after that continued washing with mercuric chloride (0.1% w/v) for 1 min. Finally, the explants were washed with 70% ethanol followed by 3 times washing with sterile distilled water, and the explants were aseptically inoculated on Murashige and Skoogs  medium supplemented with various concentrations and combinations of phytohormones for induction of callus.
The explants were cultured on MS (Murashige and Skoog)  basal medium supplemented with various concentrations of BA (Benzyl Adenine) (0.5-5.0 mg/l) + NAA (1-naphthaleneacetic acid) (0.5-5.0 mg/l) for callus induction. BA 1.0 mg/l + NAA 1.0 g/l is the best concentration for callus induction [Figure 1]. After 30 days, old callus was collected and sub-cultured on to fresh medium with same growth regulator combinations twice in 4-week time interval. All the cultures were incubated at 24 ± 2° C under 16 h photoperiod provided by cool white florescent lights.
|Figure 1: (a, b) In-vitro regenerated callus of Biophytum sensitivum after eight weeks of culture with BA 1.0 mg/l + NAA 1.0 mg/l. (c) Callus after six weeks of culture with BA 5.0 mg/l + NAA 5.0 g/l|
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Extraction from callus cultures
About 6-8 week-old calli derived from the leaf cuttings were collected and dried in an oven at 40 ± 1°C for 5 hours [Figure 1]a, b and c. Dried calli was homogenized to a fine powder and stored in airtight bottles. 25 g of leaf calli powder were extracted with 150 ml of solvent methanol for 24 h by using Soxhlet apparatus (Borosil, India). The extract was dried in a flash evaporator for 30 min, and the left over powder was considered 100%. 100 mg/ml were prepared by re-dissolving the extracted powder in the same solvent, which was used in the extraction. This crude callus extract is used for pharmacological in-vitro analyzes.
DPPH free radical scavenging activity
DPPH (1, 1-diphenyl-2picryl-hydrazyl) free radical scavenging of test compounds was determined by the method of Lamaison et al., (1991),  which depends on scavenging of colored free radical (DPPH) in methanol solution by the test drugs. The reaction mixture was prepared using DPPH and test drugs in final concentrations of 3 ml. Absorption of DPPH at its adsorption maximum 516 nm is inversely proportional to the concentrations of the scavenger (Test drug). The activity was expressed as inhibitory concentrations 50 (IC50) i.e., the concentration of the test solution showed 50% reduction in absorbance of the test solution as compared to that of blank solution.
IC50 = [(OD of control - (OD of test - OD test blank)) χ OD of control] × 100
DPPH Free radical scavenging activity inhibition was calculated using following formula.
α- glucosidase inhibition assay
α-glucosidase inhibitory activity was determined according to method of Padmanabha Rao and Jamil (2011).  In a microplate well, 50 μl of enzyme (0.4 U/ml) was taken, to this, 90 μl of 100 mM phosphate buffer pH 7.0 and 10 μl test substances was added and mixed well. The reaction mixtures was incubated at room temperature for 5 min and 50 μl of p-Nitro phenyl α-D- glucosidase (20 mM) as substrate was added, mixed well, and incubated for 15 min at room temperature. The reaction was stopped by the addition of 30 μl of sodium carbonate solution (200 mM). The absorbance was measured at 405 nm using microplate reader. Control and test blank OD's were obtained by replacing enzyme with buffer.
α- glucosidase inhibition was calculated using following formula.
Estimation of acetylcholinesterase assay
The acetylcholinesterase activity was determined using photometric method as described by Ellman et al., (1961).  Acetylthiocholine substrate is hydrolyzed by AchE in the sample and forms thiocholine, which will react rapidly and irreversibly with 56-thio-bis-nitro benzoic acid (DTNB) producing a yellow anion of 5-thio-2 nitro benzoic acid. The increase in color intensity was measured specrophotometrically at 412 nm. Twenty micro liters of 0.075 M acetylcholine iodide was added to the reaction mixture in the cuvette and mixed well and the absorbance was for 5 min at an interval of 15 seconds each, the change in absorbance per minute (∆A) was calculated (Lowry et al. 1951). 
AChE activity was calculated using following formula.
Tyrosinase inhibition assay
Tyrosinase inhibition assay was carried out according to the method of Ohguchi and Tanaka (2003).  The assay mixture contained 250 μl of enzyme, 50 μl of drug carrier, and 1250 μl of 8 mM M L - Dopa. The reaction was started by addition of substrate. Then, incubated for 1 min, the activities were measured at 475 nm. In the inhibition studies, the activities were measured in the presence of various concentrations of test substances. All the assays were performed in duplicate or triplicate.
Tyrosinase inhibition was calculated using following formula
| Results|| |
DPPH - inhibition assay
For determining the antioxidant potential (DPPH radical scavenging assays) of B. sensitivum, the stock solution (100 μg/ml) of methanol callus extract was prepared. From this stock solution, different dilutions (25, 50,100, 200 μg/ml) of extracts were prepared in methanol and water and were taken in 4 different test tubes, compared with standard vitamin - C (1, 2.5, 5, 10 μg/ml). Experiments on antioxidant potential of B. sensitivum revealed that the percentage of DPPH inhibition increased with increase in the concentration, highest percentage of inhibition was observed at 27.13 μg/ml [Figure 2]. The IC50 value of B. sensitivum was 736.5 μg/ml, indicating the less degree of in-vitro antioxidant potential of B. sensitivum when compared with control vitamin C [Figure 3]. Vitamin-C showed highest percentage of inhibition of DPPH at 160, even at 10 μg/ml.
|Figure 2: DPPH scavenging activity of B. sensitivum callus methanol extract|
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α- Glucosidase inhibition assay
The present study was to investigate in-vitro α-glucosidase inhibition at different concentrations: 37.5, 75, 150, 300 μg/ml of extract percentage of inhibition were 0.47, 4.25, 12.99, 30.02 in comparison to Acarbose (DMSO) at 10 ng/ml of using standard. The extract is found to be active at a concentration (300 μg/ml) and exhibited 30.02% inhibition. The crude extract has α- glucosidase with IC 50 value is 504.8 μg/ml [Figure 4] and [Figure 5].
|Figure 4: α-glucosidase inhibition of B. sensitivum callus methanol extract|
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Estimation of acetylcholinesterase
The acetylcholinesterase activity carried out according to the method of Ellman et al. 1961. The AChE inhibitory activity of methanol crude extract at concentrations of 100, 250, 500, 1000 ng/ml was investigated and compared to the Neostigmine (Standard) [Figure 6]. This extract possessing AChE inhibitory activity with IC 50 value is 1587 ng/ml [Figure 7] while IC50 value of standard drug is 38.93 ng/ml.
|Figure 7: Acetyl cholinesterase inhibition of B. sensitivum callus extract|
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Tyrosinase inhibition assay
Plant extracts were screened for tyrosinase inhibition assay, was carried out at 4 different concentrations of 25, 50, 100, 200 μg/ml, then the percentage of inhibition were observed respectively as 8.62, 14.39, 23.32, and 42.13% with an IC 50 values 280.5 μg/ml [Figure 8]. In comparison, the IC 50 for Resveratrol, which is used as a positive control for tyrosinase inhibition, was 8.52 μg/ml [Figure 9]. For the first time, we report tyrosinase inhibitory activity of Biophytum sensitivum.
| Discussion|| |
It is well-known that, in-vitro cultures are able to produce secondary metabolites in quantities more than that of original parts of the plants.  In the past few decades, secondary metabolite production from plant tissue culture has been identiﬁed as a tremendous resource for new drug development and clinical research in the fields of pharmacology and medicine. Callus culture is one of the main biotechnological approaches in the production of medicinal compounds from plants.  Plant cell culture extracts have also been used widely in the form of fractions and isolated compounds as potential bioactive molecules. In-vitro studies are highly instrumental in selecting a drug for a particular disease and also in getting the preliminary evidence to proceed for further in-vivo pharmacological research. Hence, the study is designed to screen and identify the therapeutic suitability of this plant extract for the treatment of a particular disease.
Therefore, in this study, antioxidant, α-glucosidase, acetylcholesterase, tyrosinase activity evaluated to determine the medicinal values. Leaf extract of Biophytum sensitivum used in traditional Nepalese folk medicine for the treatment of hyperglycemic patients.  Our in-vitro enzymes studies revealed that Biophytum sensitivum callus extract inhibits the -α-glucosidase, but the potency of this callus extract less, when compared to the standard drug, acarbose. The present study clearly indicates that the active principles in this plant extract may be helpful in preventing the glucose absorption in the gastrointestinal tract of diabetic patients. This could be an additional evidence to validate the use of this plant extract in addition to the previous findings  suggest that the hypoglycemic response of B. sensitivum may be mediated through stimulating the synthesis/release of insulin from the beta cells of Langerhans. Plant extracts were screened for tyrosinase inhibitory activity. 
Furthermore, flavonoids and other phenolic compounds have been shown to possess anti-cholinesterase properties  as observed in the case of Biophytum sensitivum callus extracts (un published data). The results elucidated that the inhibitory potential of Biophytum sensitivum against AchE was comparatively lower. This is because crude extracts contain non-active components along with active ones. Therefore, to isolate the active compounds from this plant, callus will help to identify the potent natural inhibitors of AchE. The presences of flavonoids possess anti-allergic, anti-inflammatory, anti-viral, and anti-oxidant activities. 
For the first time, we report in-vitro pharmacological screening for methanol extract of callus Biophytum sensitivum. Though callus extract has shown in-vitro inhibition of enzyme activities like α-glucosidase, acetyl cholinesterase, and tyrosinase, the potency (IC 50 ) was found to be low including with antioxidant activity. The occurrence of antioxidant activity may be due to the presence of phenolic compounds in the methanol callus extract. The reason for low antioxidant activity of B.sensitivum callus extract could be due to presence of phenol compounds in low concentration. This type of results previously reported that media with NAA concentration from 0.5 to 1.0 mg/ml had adverse effect where antioxidant activity was to be low (10-15%), as reported in the case of Ipomea aquatica leaf callus antioxidant activity. 
| Acknowledgments|| |
The first author S. Chandra Kala is thankful to University Grants Commission, New Delhi for providing financial support with Rajiv Gandhi National Fellowship. In addition, we are also grateful to the Pharmacology Department of Laila Research Centre in Vijayawada, Andhra Pradesh, India for their collaboration to carry out this work.
The opinions expressed in this publication are those of the authors and do not necessarily represent those of SCIBIOLMED.ORG. Authors are responsible for their citing of sources and the accuracy of their references and bibliographies. The editors cannot be held responsible for any lacks or possible violations of third parties' rights.
| References|| |
|1.||Shivanna MB, Vasanthakumari MM, Mangala MC. Regeneration of Biophytum sensitivum through organogenesis and somatic embryogenesis. Indian Journal of Biotechnology 2009;127-31. |
|2.||Kirtikar KR, Basu BD. Indian Medicinal Plants, vol. 3. International Book Distributors; Deharadun: 1999. p. 440-1. |
|3.||Sunil C, Latha PG, Suja SR, Shine VJ, Shyamal S, Anuja GI. Effects of ethanolic extract of Pisonia alba Span. Leaves on blood glucose levels and histological changes in tissues of alloxan induced diabetic rats. Int J Appl Res Nat Prod 2009;2:4-11. |
|4.||Puri D, Baral N. Hypoglycemic effect of Biophytum sensitivum in alloxan diabetic rabbits. Indian J Physiol Pharmacol 1998;42:401-6. |
|5.||Jachak SM, Bucar F, Karting J. Anti-inflammatory activity of extracts of Biophytum sensitivum in carrageenan induced paw edema. Phytother Res 1994;13:73-4. |
|6.||Puri D. Hypocholesterolemic Effect of Biophytum sensitivum Leaf water extract. Pharm Biol 2003;41:253-8. |
|7.||Guruvayoorappan C, Girija K. Effect of Biophytum sensitivum on B16 - 10 melanoma cells induced metastasis. Indian J Med Res 2005;13:56-9. |
|8.||Puri D, Baral N, Upadhyaya BP. Indigenous plant remedies in Nepal used in heart diseases. J Nepal Med Assoc 1997;36:334-7. |
|9.||Bhaskar VH, Rajyalakshmi V. Anti- tumor activity of aqueous extract of Biophytum sensitivum Linn. Annals of biological research 2010;1:76-80. |
|10.||Guruvayoorappan C, Afira AH, Kuttan G. Antioxidant potential of Biophytum sensitivum extract in-vitro and in vivo. J Basic Clin Physiol Pharmacol 2006;17:255-67. |
|11.||Natarajan D, Shivakumar MS, Srinivasan R. Antibacterial activity of leaf extracts of Biophytum sensitivum. Journal of pharmaceutical sciences and Research 2010;2:717-20. |
|12.||Ananda Prabu K, Kumarappan CT, Sunil Christudas, Kalaichelvan VK. Effective on Biophytum sensitivum on streptozotin and nicotinamide induced diabetic rats. Asian Pacific Journal of Tropical Biomedicine 2012;31-5. |
|13.||Dias ACP, Seabra RM, Andrade PB, Ferreres F, Ferreira M. Xanthone biosynthesis and accumulation in calli and suspended cells of Hypericum androsaemum. Plant Science 2000; 150: 93-101. |
|14.||Narkhede MB, Ajimire PV, Wagh AE, Mohan M, Shivashanmugam AT. In-vitro Antidiabetic activity of Caesalpinia digyna methanol root extract. Asian Journal of Plant Sciences and Research 2011;1:101-6. |
|15.||Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 1962;150:473-97. |
|16.||Lamaison JL, Petitjean-Freytet C, Carnat A. Medicinal Lamiaceae with antioxidant activity potential sources of rosmarinic acid. Pharm Acta Helv 1996;66:185-8. |
|17.||Rao AP, Jamil K. Pharmacological evaluation of herbal extracts for their in-vitro hypoglycemic activity, vol 3. International Journal of Pharma and Bio Sciences 2011. p. ISSN: 0975-299. |
|18.||Ellman GL, Courtney KD, Andres V Jr, Feather - Stone RM. A new and rapid colorimetric determination of acetyl cholinesterase activity. Biochem Pharmacol 1961;7:88-95. |
|19.||Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 1977;83:346-56. |
|20.||Ohguchi K, Tanaka T, Kido T, Baba K, Iinuma M, Matsumoto K, et al. Effects of hydroxystilbene derivations on tyrosinase activity. Biochem Biophys Res Commun 2003;307:861-3. |
|21.||Fugitha Y, Hara Y, Ogino T, Suga C. Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrihizon. Plant Cell Reports 1981;1:59-60. |
|22.||Krishnaraju AV, Rao TV, Sundarraju D, Vanisree M, Tsay HS., Subbaraju GV. Biological screening of medicinal plants collected from Eastern Ghats of India using Artemia salina (Brine Shrimp Test) International journal of Applied Science and Engineering 2006;4:115-25. |
|23.||Chen CH, Chan HC, Chu YT, Ho HY, Chen PY, Lee TH, et al. Antioxidant Activity of Some Plant Extracts Towards Xanthine Oxidase, Lipoxygenase and Tyrosinase Molecules 2009;14:2947-58. |
|24.||Orhan I, Kartal M, Tosum F, Sener B. Screening of various phenolic acids and flavonoid derivatives for their anticholinesterase potential. Z Naturoforsch C 2007;62:829-32. |
|25.||Ji HF, Zhang HY. Multipotent natural agents to combat Alzheimer's disease. Functional spectrum and structural features. Acta Pharmacol Sin 2008;29:143-51. |
|26.||Anand Prasad K, Siva Prasad M, Shivamurthy GR, Aradhya SM. Callus induction from Ipomoea aquatic Forsk Leaf and its antioxidant activity. Indian Journal of Biotechnology 2006;:107-11. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
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