Evaluation of the Effect of Salicylic Acid on Production of Flavonoid and Phenolic content of Leaves Extract of Gardenia gummifera

 

Rajiv Saxena¹*, Neelesh Malviya^{1, 2}

¹Mandsaur University, Rewas Dewda Road, SH - 31, Mandsaur, Madhya Pradesh- 458001

²Smriti College of Pharmaceutical Education, MR-11, Dewas Naka, Indore, Madhya Pradesh-452010

 

ABSTRACT:

Herbal drugs treatment has covered a wide spectrum disorders under its umbrella of it and industrial and technological advancement makes these drugs more accurate and valued. Overexploitation of rich flora, factors like global warming, deforestation, lack of availability and awareness makes these drug sources in the erg of extinction. Many newer approaches including the use of elicitors are coming up to fight with the situation and scientists are coming up with latest solutions. Gardenia gummifera belonging to the family rubiaceae is one such plant species which has many pharmacological benefits but again the plant is about to extinct. The present investigation was focused on the use of selected Elicitors Salicylic acid (SA) for the enhanced production of phytochemicals of Gardenia gummifera cultivated under controlled environmental conditions in polyhouse developed in medicinal garden of the Institute. The plants were exposed to Salicylic acid (SA) as an elicitor at various concentration 0.50, 0.100 and 0.150 mg/l and the total flavonoid content was calculated as Quercetin equivalent (mg of Quercetin /g of extract) (QE) and was found to be 22.45, 45.24, 37.93 whereas for the untreated plants 15.2 QE was obtained. The phenolic content was calculated as Gallic acid equivalent (mg of gallic acid /g of extract) (GAE) and was found to be 23.29, 39.53, 38.54 whereas for the untreated plants 18.36 GAE was obtained. In both the cases the results suggested that SA exposure enhances flavonoids and phenolic content production in Gardenia gummifera at a concentration of 0.100 mg/litre.

 

 

INTRODUCTION:

The plant derived medicines are getting popularity, not only in developing countries but also in developed countries. The reason behind is the safety of drugs and none or minimal side effects, effectiveness in almost all the diseases and the low and affordable cost. When we explore ethnopharmacological evidences, we find that traditional medicines are widely used as a remedy for the treatment in dreadful diseases from ages. India is always been the most popular country as far as the medicinal plant research is concerned. Many big Pharmaceutical Industries are being set up that are engaged in Herbal medicine product manufacturing. Modern applications of technology on herbal medicines have developed novel drug delivery system for the better drug delivery^1,2.

 

 

The application of nano technology in formulations of herbal drugs are reported to have advantages over conventional crude drugs preparations and enhancement of pharmacological activity and solubility of herbal drugs, reduced toxicity, sustained drug delivery etc.³

 

The strive to good health with the help of herbal supplements is trending all over the world. The focus of many Research and Development groups are now concentrated on herbal drugs and many new formulations with many combinations of herbal drugs are flourished in the market. The consequence of such over exploitation of rich flora from natural habitat limits the free flowing supply of crude drug. Not only this, but many manmade factors like global warming, climate change, pollution etc. are effecting the human health⁴. They also constrict the availability of these sources of medicines and affecting the human health. The overall impact of such condition is the extinction of many plant varieties from our nature. The plant used in the present study was Gardenia gummifera and it also marked as endangered plant species⁵. Gardenia gummifers belongs to the family Rubiaceae and its a small tree. The average height of the plant is about 3-7 meters. Flowers are bisexual and white in color. Studies on pharmacognostic characteristics of plant leaves showed that epidermal cells are predominantly 5-7 sided and few are 4 sided as well and mostly polygonal anisodiametric to isodiametric⁶. The resin exuded from the plant has many pharmacological activities like wound healing, anti-ulcer, cardio vascular disorders etc. The plant major phyoconstituents of the plant includes Cycloartane triterpenes i.e dikamaliartanes, flavonoids including 3′, 5, 5′-trihydroxy-4′, 6, 7, 8-tetramethoxyflavone⁷, gardenin A-E along with aldehydes, mannitol, sitosterols etc.⁸ The phenolic and flavonoid content are involved in many pharmacological activities including antioxidant activity⁹. Elicitor technology is one way to enhance the production of phytopharmaceuticals specially when we keep in mind the medicinal plants which are red marked or in the erg of extermination. Elicitors basically work by triggering plant defence mechanism and may result in the enhanced production of various phyochemicals or phytoalexins, Pathogen related (PR) proteins and many other defensive molecules as shown in figure 1A and 1B. It has been observed in many studies that salicylic acid as an elicitor enhances the production of plant secondary metabolites¹⁰. Like salicylic acid many abiotic elicitors are found to increase the production of phtoconstituents in many medicinally important plant like withania somnifera¹¹. The plant under investigation was cultivated in controlled environmental condition in green-house and different concentrations of Salicylic acid (SA) were sprayed on plant at different time interval. After the exposure of 40 days the leaves of the plant was collected and successive solvent extraction with petroleum ether, chloroform, methanol and water was done using soxhlet apparatus. The phyochemical screening tests for the presence of different phytochemicals was performed. The total flavonoid and phenolic content was determined in each extract.

 

Figure 1A and 1B showing the comparative effect of plant pathogen and elicitor

 

MATERIAL AND METHODS:

Gardenia gummifera was collected from poly house of Smriti college of Pharmaceutical Education, Indore. Salicylic acid, Petroleum ether, Methanol, mayer’s reagent, dragendroff reagent, hager’s reagent, wagner’s reagent, molish reagent, fehling’s A and B, ferric chloride, lead acetate, bromine solution, ninhydrin reagent, HCl, Zinc dust, acetic anhydride, sulphuric acid, benzene, sodium hydroxide, quercetin, Acetic Acid. Folin-Ciocalteu reagent, Sodium carbonate.

 

Construction of poly-house at Institute:

The poly house was constructed in the medicinal garden of Institute to cater the suitable and controlled environmental condition for the growth of plant under study (Gardenia gummifera). The designing of poly-house was done using natural materials like bamboo for the easy growth of plant in natural habitat like conditions. The poly-house was having the proper facility of irrigation and drainage.

 

Cultivation of gradenia gummifera in poly house and treatment with selected elicitor:

The healthy plants were cultivated in the constructed poly-house after the authentication of plant by Dr. S.D. Upadhyaya, Professor & Head, Department of Forestry and Medicinal Plants, J.N.K.V.V, Jabalpur. in randomized block designing method as shown in figure 2. The poly-house was designed in such a way that separate division was allotted for the growth of elicitor (SA) treated plant, and control group. All the plants were well irrigated regularly using drip system. Within 9-10 days of acclimatization the healthy plants showed the growth signs and apical leaves development were visible. The selected elicitor (SA) was than sprayed using spray bottles with different concentration of salicylic acid (SA) at the region of treated plants division. The untreated group also was taken care for their growth and regular irrigation was done. The elicitor treatment was continued for 45 days at regular interval of time.

 

SA (0.100 mg/l conc)	SA (0.150 mg/l conc)	SA (0.50 mg/l conc)	Free moving space	Untreated plants of Gardenia gummifera
SA (0.150 mg/l conc)	SA (0.50 mg/l conc)	SA (0.100 mg/l conc)		Untreated plants of Gardenia gummifera
SA (0.50 mg/l conc)	SA (0.100 mg/l conc)	SA (0.150 mg/l conc)		Untreated plants of Gardenia gummifera

Figure 2: Distribution of plants in poly house

 

Collection, Drying and extraction of leaves of gardenia gummifera:

After the completion of 45 days of elicitor treatment the leaves of Gradenia gummifera was collected and allowed to shade dry shattered and screened with the help of 40 no. mesh. It was soxhlet extracted with petroleum ether for defatting for 4hr at 60^oC. After drying and levigation the marc was extracted with methanol using soxhlet apparatus. The percentage yield was calculated.

 

Phytochemical Examination of Extract:

Phytochemical identification tests was performed in the extract of elicitor (SA) treated plants and untreated plants as per the standard methods for the qualitative analysis of Phenolic compounds & tannins, Alkaloids, Saponins, Proteins & Amino acids, Flavonoids, Phytosterols, Terpenoids, Carbohydrates & glycosides, gums^{12, 13}.

 

Determination of Total Flavonoid Content:

The determination of total flavonoid content was done by aluminium chloride method using colorimetric assay^14,15. The assay was performed in triplicate. The 0.5 ml. solution of extract of Gardenia gummifera in methanol (5mg/100ml.) was mixed with 2 ml. distilled water and 150 µl of 5% sodium nitrate. The resultant mixture was kept for 6 minutes and than 150 µl of 10% aluminum chloride and 2mL of 1 M sodium hydroxide was added and kept at room temperature for another 15 minutes.

 

The Absorbance of the solution was measured by UV-Visible spectrophotometer at 510 nm and total flavonoid contents were calculated as quercetin equivalents from a calibration curve of quercetin.

 

Determination of total Phenolic content:

The amount of phenolics in methanolic extract of Gardenia gummifera was determined with the help of Spectrophotometric method using Folin-Ciocalteu reagent^{16, 17}. The assay was performed in triplicate. Added 2.5 ml of 10% Folin-Ciocalteu reagent and 2 ml of Na₂CO₃ (7.5% w/v) to 0.5 ml of each sample of methanolic extract of plant under investigation (1mg/ml). The blank was also prepared by admixing 0.5 ml of methanol 2.5 ml Folin-Ciocalteu’s reagent (10%, Prepared in water) and 2.5 sodium carbonate (7.5%). The resulting mixture was incubated at 45°C with constant shaking for 15 min. The absorbance of the samples was measured at 765 nm using spectrophotometer. The content of total phenol was calculated on the basis of the calibration curve of gallic acid and the results were expressed as mg of gallic acid equivalents (GAEs) per gram of extract.

 

Statistical analysis of data:

The data were analysed using Microsoft Excel and reported as mean ± SEM of triplicate determination.

 

RESULTS AND DISCUSSION:

Analysis of pharmacognostic activities of leaves of Gardenia gummifera:

Table 1: Organoleptic features of dried leaves of Gardenia gummifera

Common Name of plant	Scientific name	Part of the Plant Used in the study	Colour	Odour	Texture
Dikamali	Gardenia gummifera	Leaves	Dark green	Pleasant	Granular

 

Table 2: Percentage yield of methanolic extract of leaves of Gardenia gummifera

Name of solvent	Percentage yield (Untreated plants)	Percentage yield (Treated with 0.50 mg/l SA)	Percentage yield (Treated with 0.100 mg/l SA)	Percentage yield (Treated with 0.150 mg/l SA)
Methanolic Extract	11.52	10.15	9.57	11.40

 

Table 3: Phytochemical qualitative tests of extract

 

 

 

Quantitative Analysis of Extract for its Flavonoid content:

 

Figure 3: Standard curve of Quercetin

 

Table 4: Total Flavonoid Content of the plant extracts (Methanolic extract of Gardenia gummifera) expressed in terms of Quercetin equivalent (mg of Quercetin/g of extract) subjected to elicitor application (SA):

S. No.	Concentration of Elicitor (SA) (mg/l)	Quercetin equivalent (mg of Quercetin /g of extract) (QE)
1	0.50	22.45±0.076
2	0.100	45.24±0.083
3	0.150	37.93±0.065
4	Untreated plant (Control)	15.2±0.057

 

Figure 4: Total flavonoid content at various concentration of Salicylic acid application

 

Figure 5: Standard curve of gallic acid

Table 5: Total Phenolic Content of the plant extracts (Methanolic extract of Gardenia gummifera) expressed in terms of gallic acid equivalent (mg of GA/g of extract) subjected to elicitor application (Salicylic acid):

S. No.	Concentration of Elicitor (SA) (mg/l)	Gallic acid equivalent (mg of GA/g of extract) (GAE)
1	0.50	23.29±0.092
2	0.100	39.53±0.008
3	0.150	38.54±0.011
4	Untreated plant (Control)	18.36±0.005

 

Figure 6: Total phenolic content at various concentration of Salicylic acid application

 

In the present study methanolic extracts of elicitor (SA) treated plants Gardenia gummifera at various concentrations was analysed. The percentage yield of untreated and 0.50, 0.100 and 0.150mg/l salicylic acid treated plant was found to be 11.52, 10.15, 9.57 and 11.40 respectively.

 

The phytochemical screening of the extract shows the presence of tannins, glycosides, Phenolic compounds, Flavonoids, phyosterols, Terpenoids (Table-3).

 

The total flavonoid content was calculated as Quercetin equivalent (mg of Quercetin/g of extract) (QE) at different concentration of SA as 0.50, 0.100 and 0.150 mg/l and was found to be 22.45, 45.24, 37.93. The total flavonoid content in the untreated plants was found to be 15.2. The result suggests that the total flavonoid content was maximum at concentration 0.100mg/l SA treated plants and shows enhancement as compare to untreated plant with 15.12 mg of Quercetin/g of extract.

 

The total phenolic content was calculated as Gallic acid equivalent (mg of GA/g of extract) (GAE) at different concentration of SA as 0.50, 0.100 and 0.150mg/l and was found to be 23.29, 39.53, 38.54. The total phenolic content in the untreated plants was found to be 18.36. The result suggests that the total phenolic content was maximum at concentration 0.100 mg/l SA treated plants and shows enhancement as compare to untreated plant with 18.52mg Gallic acid /g of extract.

 

The stress conditions in plants are regulated by several defensive mechanisms. Phytoconstituents of the plant also plays a major role along with pathogen related proteins etc. The triggering of these responses occurs as soon as the plant undergoes stress, may be due to the attack of plant pathogen. Medicinally important plant can be artificially stresses by the use of elicitors and hence, elevated level of phytochemicals or sometimes generation of new metabolite can be achieved. Gardenia gummifera is a red marked plant i.e at the erg of extinction. So, in the present study an approach to increase the level of flavonoid and phenolic content by the use of salicylic acid as an elicitor in various concentrations (0.50mg/l, 0.100mg/l, 0.150mg/l) was done. Elicitors specially salicylic acid enhances the production of phytochemical compounds¹⁸ in response to biochemical stress. Different abiotic stresses triggers the production of enzymes which are involved in Salicylic acid biosynthesis and thus help the plant to fight against many environmental stresses¹⁹. Phenolic and flavonoid compounds are specially respond to increase with Salicylic acid treatment. The probable mechanism responsible for the activity is the induced production of hydrogen peroxide by salicylic acid in turns stimulate the enzyme phenylalanine ammonium lyase which is responsible for the generation of phenolic compounds²⁰.

 

CONCLUSIONS:

Ayurvedic system of medicine have proven potential in the management of diseases from ages. Herbs are very popular source of medicine and even in the modern era of treatment, people are striving them to achieve good health. The scientific knowledge of plant responses to the environment and stress had opened a new vista of research by the use elicitors that can enhances the generation of plant secondary metabolites specially to the endangered plant species. Gradenia gummifera belongs to the family rubiaceae is labelled as one such plant that is at the edge of extinction from natural habitat. In the present study it is been observed that the application to Salicylic acid as an elicitor enhances the level of flavonoids and phenolic content. Further the chemical characterization is needed to qualitatively identify the specific flavonoids and phenolic compounds and also the analysis of other generated phytochemicals is required to get the knowledge of complete effect of SA on Gradenia gummifera.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Sanghi DK, Tiwle R. Herbal Drugs an Emerging Tool for Novel Drug Delivery Systems. Research J. Pharm. and Tech. 2013; 6(9): Page 962-966.

2.      Rinku Y. Pati, Shubhangi A. Patil, Niranjan D. Chivate, Yogesh

3.      N. Patil. Herbal Drug Nanoparticles: Advancements in Herbal Treatment. Research J. Pharm. and Tech. 2018; 11(1): 421-426.

4.      Yogita R. Indalkar, Nayana V. Pimpodkar, Anita S. Godase, Puja S. Gaikwad. A Compressive Review on the Study of Nanotechnology for Herbal Drugs. Asian J. Pharm. Res. 5(4): 2015; 203-207.

5.      Sankranti Queen Mary. Impact of Global Warming on Health. Int. J. Nur. Edu. and Research. 2017; 5(4): 370-376.

6.      Gajakosh AM, Jayaraj M, Sonappanavar AB, et al. In vitro morphogenic response of leaf and shoot tip explants of Gardenia gummifera (l.) f.-a medicinal plant. International Journal of Pharmacy & Technology. 2011; 3(2): 2487-2495.

7.      Sudhakar P, Ramachandra Reddy P. Pharmacognostic and HPTLC Finger printing of Gardenia gummifera L.F. Res. J. Pharmacognosy and Phytochem. 2017; 9(4): 235-240.

8.      Kunert O, Sreekanth G, Babu GS. Rao, BVRA. Radhakishan, M. Kumar B.R., Saf R, Rao AVNA, Schühly W. Cycloartane triterpenes from dikamali, the gum resin of Gardenia gummifera and Gardenia lucida. Chem. Biodivers. 2009; 6: 1185–1192.

9.      Reddy GC, Rangaswami S, Sunder R. Triterpenoids of the stem bark of Gardenia gummifera. Planta Med. 1977; 32: 206-211.

10.   Gahlot K, Lal VK, Jha S. Total Phenolic content, flavonoid content and In vitro antioxidant activities of Flemingia species (Flemingia chappar, Flemingia macrophylla and Flemingia strobilifera). Research J. Pharm. and Tech. 2013; 6(5): 516-523.

11.   Hayat S, Irfan M, Wani A, Nasser A, Ahmad A. Salicylic acids: Local, systemic or inter-systemic regulators?. Plant Signal Behav. 2012; 7: 93–102.

12.   Kaur P, Tiwari A. Use of Abiotic Elicitors to enhance Secondary Metabolites Production in Withania somnifera. Research J. Pharm. and Tech. 2018; 11(11): 5139-5142.

13.   Brain KR, Turner TD. The practical evaluation of phytopharmaceuticals, Bristol: Wright Sciencetechnica.1975. pp. 4-9.

14.   Evans WC. Trease and Evans Pharmacology, WB Saunders Company Ltd. 2000; 4th ed: pp. 224- 239.

15.   Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry. 1999; 64 : 555-559.

16.   Sheela D, Cheenickal M. Total Phenolics and Flavonoids among the Selected Species of Syzygium, Gaertn. Res. J. Pharmacognosy and Phytochem. 2017; 9(2): 101-104.

17.   Singleton VL, Orthofer R, Lamuela Raventos RM, Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Methods Enzymol.1999; 299: 152-178.

18.   Parimi R, Pravallika KE. Studies on Phytochemical Screening, Total Phenolic Content and In vitro Antioxidant activity of Different Extracts of Euphorbia thymifolia Roots. Research J. Pharm. and Tech. 2017; 10(2): 551-556.

19.   El-Gaied LF, Abu El-Heba GA, El-Sherif NA. Effect of growth hormones on some antioxidant parameters and gene expression in tomato. GM Crops Food. 2013; 4:67–73.

20.   Aziz A, Kapoor D. Salicylic Acid: It’s Physiological Role and Interactions. Research J. Pharm. and Tech. 2018; 11(7): 3171-3177.

21.   Hao W, Guo H, Zhang J, Hu G, Yao Y, Dong J. Hydrogen peroxide is involved in salicylic acid-elicitedrosmarinic acid production in Salvia miltiorrhiza cell cultures, The Scient. World J. 2014; 2014: 843764.

 

 

 

 

Received on 14.06.2019           Modified on 21.07.2019

Accepted on 28.08.2019         © RJPT All right reserved