Comparative physicochemical, phytochemical and high performance thin layer chromatography evaluation of heart wood and small branches of Pterocarpus marsupium

Bhavana Srivastava¹*, Himanshu Sharma¹, Vikas Chandra Sharma¹, S.C. Verma², A.D. Jadhav¹

¹National Research Institute for Ayurveda Siddha Human Resource Development, Aamkho, Gwalior-474009, (M.P), India.

²Pharmacopeia Commission for Indian Medicine & Homeopathy, PLIM Campus, Kamala Nehru Nagar, Ghaziabad-201002.

*Corresponding Author E-mail: bhavanakan@gmail.com

ABSTRACT:

Pterocarpus marsupium commonly called Indian kino tree is a medicinal plant widely used in Ayurveda. As per the Ayurvedic literature, heart wood of this plant is used in Krmiroga, Kustha, Prameha, Pandu, and Medodosa. Removal of heart wood from trunk of this tree may make this plant weak and susceptible to damage by insects and natural elements.Due to which availability of this plant may be difficult in near future for use in Indian system of medicine. This work is an attempt to evaluate the possibilities of using small branches in place of heart wood. The standard parameters of small branches of P. marsupium have not been prepared yet. So work is carried out to establish preliminary physicochemical and phytochemical standards of small branches of P. marsupium. Heart wood and small branches of P. marsupium are compared on the basis of physicochemical analysis, phytochemical analysis and high performance thin layer chromatography (HPTLC). Total phenolic contents of heart wood and small branches in terms of tannic acid equivalent were 36.65±0.90 and 41.91±1.05 mg/g, respectively and total flavonoid contents in terms of querecetin equivalent were 56.30±0.38 and 70.22±1.25 mg/g, respectively. Phytochemical analysis of heartwood and small branches showed the presence of phenols, tannins, alkaloids, carbohydrates, saponins, proteins, steroids, flavanoids, coumarin, quinine and furanoids in various extracts tested. HPTLC of n-hexane, ethyl acetate and ethanol extracts of heart wood and small branches showed different phytochemical profile. Difference in HPTLC profiles suggests that small branches cannot be used in place of heart wood and further research is required to find out the substitute for heart wood of P. marsupium. Study will be helpful in the identification and quality control of P. marsupium and can provide standard HPTLC profiles of P. marsupium with selected solvent system for use as a reference for the proper identification/ authentication of the drug. Good amount of total phenolics and total flavonoids in small branches and HPTLC profile with many bands indicates that small branches may also have potential active constituents and may be studied for various pharmacological activities.

KEYWORDS: Pterocarpus marsupium, physicochemical analysis, phytochemical analysis, HPTLC profile.

INTRODUCTION:

Asia represents one of the most important centers of knowledge with regard to use of medicinal plants in providing health care for both humans and animals not only in diseased condition but also as potential material for maintaining proper health. There are many medicinal plants which are slow-growing forest trees and heart wood of which is part mainly utilized. Removal of heart wood from trunk of these trees makes the plant weak and susceptible to damage by insects and natural elements. To safeguard the survival of these plants and to make them available for future generation usages of heart wood of trunk are prohibited. Because of this manufacturers and dealers of Ayurveda, Siddha and Unani drugs face trouble in getting regular supply of heart wood of big trees. Therefore, to ensure the availability of heartwood as raw material, there is strong need to explore the possibility of substitution of heartwood of the trunk with suitable alternate. An approach which would satisfy the necessities of sustainable harvesting, yet simultaneously provide for health care needs, would be the substitution of heart wood or underground parts with aerial part of the same plant.

Pterocarpus marsupium (Family: Fabaceae) commonly called Indian kino tree (Asana) is a medicinal plant widely used in Ayurveda.

As per Ayurvedic literature, heart wood of this plant is used in krmiroga, kustha, prameha, pandu, medodosa¹. It is also reported for cardio tonic activity², alloxan-induced diabeties³, glucose tolerance effect⁴, hypoglycemic activity ^5,6, antihyperlipidemic effect⁷, gastric ulceration⁸, cytokine TNF-α in type 2 diabeties⁹, anti-hyperglycemic activity¹⁰, free radical scavenging activity¹¹, antiallergic, antianaphylactic and mast cell stabilizing activity¹². Heart wood of this plant mainly contains pterostilbene¹³, marsupsin, liquiritigenin, isoliquiritigenin, pterosupin, p-hydroxyl benzaldehyde, 7, 4′-dihydroxyflavone, (2R)-3-(p-hydroxyphenyl) lactic acid¹⁴, propterol¹⁵, marsupol¹⁶, carpusin¹⁷, C-glucoside, 1-(2',6'-dihydroxyphenyl)-β-D-glucopyranoside¹⁸, C-glucosides, pteroside, pteroisoauroside, marsuposide, flavon C-glucoside, vijayosin, C-β-D-glucopyranosyl-2,6-dihydroxyl benzene and sesquiterpene¹⁹. Removal of heart wood from trunk of this tree may affect the survival of this plant due to which availability of this plant may be difficult in near future for use in Indian system of medicine. Present study is an attempt to evaluate the possibilities of using small branches in place of heart wood. The standard parameters of small branches of P. marsupium have not been prepared yet. So work is also carried out to establish preliminary physicochemical and phytochemical standards of small branches.

MATERIAL AND METHODS:

Plant material

The heart wood and small branches of P. marsupium were collected from NVARI, Jhansi (U.P.) India, identified and authenticated by the botanist of NRIASHRD, Gwalior.

Instrumentation

A CAMAG HPTLC system (Muttenz, Switzerland) equipped with a semi automatic TLC applicator Linomat IV, twin trough plate development chamber, Win CATS software version 1.4.2 and Hamilton (Reno, Nevada, USA) Syringe (100 μl).

Material and reagents

All chemicals, reagents and solvents used during the experiments were of analytical grade and HPTLC plates were purchased from E. Merck Pvt. Ltd. (Mumbai, India).

Physicochemical parameters

Heart wood and small branches were studied for various physicochemical standards like foreign matter, loss on drying at 105°C, total ash, acid-insoluble ash, alcohol soluble extractive, water soluble extractive and pH 10% solution using standard methods^{20, 21}.

Preliminary phytochemical screening

n-Hexane, ethyl acetate and ethanol extract of both heart wood and small branches were screened for the presence of phenols, tannins, carbohydrates, saponins, alkaloids, proteins, flavonoids, phytosterols, furanoids, coumarin and quinone by the standard methods of Harbone²⁰and Kokate²¹_.

Estimation of total phenolic and flavonoid content

Five grams of each of the shade-dried plant material was pulverized into coarse powder and subjected to ethanolic extraction using soxhlet apparatus. The extracts were concentrated to dryness. The dried residues were then dissolved in 100 ml of 95% ethanol. The extracts were used for total phenolic and flavonoid assay.

The total phenolics content was determined by using the Folin-Ciocalteu assay²². An aliquot (1 ml) of extracts or standard solution of tannic acid (20, 40, 60, 80 and 100 μg/ml) was added to a 25 ml volumetric flask, containing 9 ml of distilled water. A reagent blank was prepared using distilled water. One millilitre of Folin-Ciocalteu phenol reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na₂CO₃ solution was added to the mixture. The volume was then made up to the mark. After incubation for 90 min at room temperature, the absorbance against the reagent blank was determined at 550 nm with an UV/Vis spectrophotometer. Total phenolics content was expressed as mg tannic acid equivalents (TAE).

Total flavonoid content was measured by the aluminum chloride colorimetric assay²³. An aliquot (1 ml) of extracts or standard solutions of quercetin (20, 40, 60, 80 and 100 μg/ml) was added to a 10 ml volumetric flask containing 4 ml of distilled water. To the flask, 0.30 ml of 5% NaNO₂ was added and after 5 min, 0.3 ml of 10% AlCl₃ was added. After 5 min, 2 ml of 1M NaOH was added and the volume was made up to 10 ml with distilled water. The solution was mixed and absorbance was measured against the blank at 510 nm. The total flavonoid content was expressed as mg quercetin equivalents (QE).

HPTLC profiles

HPTLC studies were carried out following the method of Sethi²⁴, Stahl²⁵ and Wagner et al²⁶. The heart wood and small branches were powdered coarsely. Ten gram powdered samples of each of heart wood and small branches were accurately weighed and exhaustively extracted by n-hexane, ethyl acetate and ethanol (each 100 ml) separately using soxhlet apparatus. The extracts were filtered and concentrated under reduced pressure and made up to10 ml in standard flasks separately.

The mobile phase used for developing the n-hexane, ethyl acetate and ethanol extracts of heart wood and small branches was toluene: ethyl acetate 7:3 (v/v).

The samples were spotted in the form of bands of width 10 mm with a 100 μl Hamilton syringe on aluminum TLC plates pre-coated with Silica gel 60 F₂₅₄ of 0.2 mm thickness with the help of TLC semi-automatic applicator Linomat IV attached to CAMAG HPTLC system, which was programmed through Win CATS software version 1.4.2. 10 μl of each extracts of heart wood and small branches were applied in two tracks as 10 mm bands at a spraying rate of 10 seconds/μl. Track 1 was heart wood and track 2 was small branches for each of the extracts applied.

Development of the plate up to a migration distance of 80 mm was performed at 27 ± 2°C with mobile phase for each extracts in a CAMAG HPTLC chamber previously saturated for 30 min. After development the plate was dried at 60°C in an oven for 5 min and visualized under wavelength 254 nm and 366 nm for ultra violet detection. The developed plate was then dipped in anisaldehyde sulphuric acid reagent for derivatization and dried at 105°C in hot air oven till the colour of the band appears and visualized under white light. Images were captured by keeping the plates in photodocumentation chamber and R_f values were recorded by Win CATS software.

RESULTS AND DISCUSSION:

Physicochemical parameters like foreign matter, loss on drying at 105º C, ash values, acid insoluble ash, extractive values and pH values are given in Table 1. These data can be used for identification of the drug. Both the parts of P. marsupium were found to possess little moisture and hence can be stored at room temperature without fear of spoilage. Approximately same value for alcohol soluble and water soluble extractives for both heart wood and small branches indicates the presence of approximately same amount of polar and non polar extractable compounds in heart wood and small branches.

Table 1: Physicochemical parameters of heart wood and small branches of P. marsupium.

S. No.	Parameters	Results
S. No.	Parameters	Heart wood	Small branches
1.	Foreign matter (% w/w)	Nil	Nil
2.	Loss on drying (% w/w)	6.478	6.863
3.	Total ash (% w/w)	1.874	1.484
4.	Acid insoluble ash (% w/w)	0.435	0.427
5.	Alcohol soluble extractive value (% w/w)	7.167	6.66
6.	Water soluble extractive value (% w/w)	6.50	5.338
7.	pH of 10 % aqueous solution	7.72	7.41

The results of phytochemical analysis of different extracts of heart wood and small branches are shown in Table 2.

Table 2: Phytochemical analysis of extracts of heart wood and small branches of P. marsupium.

Phytochemicals	Heart wood			Small branches
Phytochemicals	n-Hexane	Ethyl acetate	Ethanol	n-Hexane	Ethyl acetate	Ethanol
Phenols	-ve	-ve	+ve	-ve	+ve	+ve
Tannins	-ve	-ve	+ve	-ve	+ve	+ve
Alkaloids	-ve	+ve	+ve	-ve	+ve	+ve
Carbohydrates	-ve	+ve	+ve	-ve	+ve	+ve
Saponins	-ve	-ve	-ve	-ve	-ve	-ve
Proteins	+ve	+ve	+ve	+ve	+ve	+ve
Steroids	-ve	-ve	+ve	-ve	-ve	+ve
Flavanoids	-ve	-ve	+ve	-ve	+ve	+ve
Coumarin	-ve	-ve	+ve	-ve	-ve	+ve
Quinone	-ve	+ve	+ve	-ve	+ve	+ve
Furanoids	-ve	-ve	+ve	-ve	+ve	+ve

Proteins were found to be present in hexane extract of both heart wood and small branches. In ethyl acetate extract alkaloids, carbohydrates, proteins and quinone were found present in both heart wood and small branches whereas phenols, tannins flavonoid and furanoids were found present only in small branches. In ethanol extract alkaloids, phenols, tannins, carbohydrates, proteins, steroids, flavanoids, coumarin, quinone and furanoids were found present in both heart wood and small branches.

Total amount of phenolics and flavonoids content of ethanoilc extract of heart wood and small branches of P. marsupium are summarized in Table 3. Results indicate that in comparison to heart wood, small branches had the high total phenolic and flavonoid content.

Table 3: Total phenolic and total flavonoid content of ethanolic extracts of heart wood and small branches of P. marsupium

S. No.	Plant parts	Total phenolics mg of TAE/g dry weight*	Total flavonoids mg of QUE/g dry weight*
1.	Heart wood	36.65±0.90	56.30±0.38
2.	Small branches	41.91±1.05	70.22±1.25

*Values are expressed as Mean ± SD

Comparative HPTLC profile of n-hexane, ethyl acetate and ethanol extracts of heart wood and small branches of P. marsupium were recorded to reveal the chemical pattern of each extract. The HPTLC profile of n-hexane extract of both heart wood and small branches (Figure 1 and Table 4) showed no band when visualized under UV at 254 nm. At UV 366 nm, heart wood and small branches showed two and three bands, respectively out of which only one band at R_f 0.63 (florescent blue) found similar. Visualization under white light after derivatization with anisaldehyde sulphuric acid reagent both heart wood and small branches showed four bands but no band found similar. This indicates the presence of different compounds in hexane extract of heart wood and small branches.

Table 4: R_fvalue of n-hexane extract of P. marsupium.

S. No.	Wavelength	Heart wood	Small branches
1.	254 nm	No band	No band
2.	366 nm	0.63, 0.69	0.63, 0.68, 0.75
3.	Visible light after derivatization	0.44, 0.51, 0.59, 0.87	0.43, 0.50, 0.62, 0.86

The HPTLC profile of ethyl acetate extract of heart wood and small branches (Figure 2 and Table 5) showed three and two bands, respectively at UV at 254 nm and no bands found similar. At UV 366 heart wood and small branches showed eight and four bands, respectively and no band found similar. Visualization under white light after derivatization with anisaldehyde sulphuric acid reagent, heart wood and small branches showed three and seven bands, respectively again none found similar. These results show the presence of different compounds in ethyl acetate extract of heart wood and small branches also.

Table 5: R_fvalue of ethyl acetate extract of P. marsupium.

S. No.	Wavelength	Heart wood	Small branches
1.	254 nm	0.27, 47, 0.61	0.09, 0.15
2.	366 nm	0.05, 0.19, 0.27, 0.39, 0.47, 0.61, 0.69, 0.74	0.10, 0.21, 0.63, 0.69
3.	Visible light after derivatization	0.48, 0.52, 0.87	0.08, 0.14, 0.21, 0.47, 0.51, 0.29, 0.86

CONCLUSION:

Present study carried out in P. marsupium will be helpful in the identification and quality control of the drug and can provide standard HPTLC profiles with selected solvent system. The HPTLC profile can also be used as a reference for the proper identification/ authentication of the drug. Difference in HPTLC profiles of n-hexane, ethyl acetate and ethanol extracts of heart wood and small branches suggests that small branches cannot be used in place of heart wood and further research is required to find out the suitable substitute for heart wood of P. marsupium. Good amount of total phenolics and total flavonoids in small branches and HPTLC profile with many bands indicates that small branches may also have potential active constituents and may be studied for various pharmacological activities.

ACKNOWLEDGEMENT:

Authors are thankful to CCRAS, New Delhi for financial assistance and Director General, CCRAS, New Delhi for providing facilities.

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Received on 04.01.2016 Modified on 10.01.2016

Res. J. Pharmacognosy and Phytochem. 2016; 8(2): 53-59

DOI: 10.5958/0975-4385.2016.00010.8