Proximate Analysis and Preliminary Phytochemical Standardization of Leaves of Two Malvaceous Plants

Switi Gaikwad*, Dr. G. Krishna Mohan and Kavitha Reddy

Centre for Pharmaceutical Sciences, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad-500 085

ABSTRACT:

Plants from Malvaceae family are also known as a ‘Mallow plants'. Two plants from this family have been selected for the present study viz. Abutilon indicum Linn. (Sweet) [AI] and Thespesia populnea Linn. (Soland) [TP]. The present study attempts to summarize the proximate analysis like Total ash, Extractive value, Foreign organic matter, Moisture content determination as well Preliminary phytochemical determination by some chemical tests of methanolic extracts of Abutilon indcium [MEAI] and Thespesia populnea [METP]. Standardization of plant extracts by different methods like Thin Layer Chromatography (TLC), High Performance Thin Layer Chromatography (HPTLC) and determination of functional group present in the crude powdered drug by Infrared (IR) Spectroscopy were carried out. Proximate composition of leaves showed that it contains total ash values of AI and TP (10 %w/w and 7 %w/w). Water soluble ash was found to be more (5 %w/w and 6.1 %w/w) than acid insoluble ash (1 %w/w and 0.64 %w/w). Flavonoids, triterpenoids, steroids, tannins, and phenolic compounds were present in the methanolic extract of the both plants. Presence of these phytochemicals were supported by Thin layer Chromatography. Total phenolic content of MEAI and METP was found to be 40.20 µg/mg and 59.88 µg/mg respectively. Total flavonoid content of MEAI and METP was found to be 7.86 µg/mg and 10.54 µg/mg respectively. HPTLC profile shows characteristic bands under UV 254 nm which helps to aid in identification of phytoconstituents, adulteration, and standardization of plant materials. IR spectrum shows presence of -OH, C=O groups with sharp peak and aromatic ring and alkanes with weak signals. So, evaluation of plant materials by these above mentioned parameters can be helpful for authentication, identification and quality plant products.

KEYWORDS: Proximate Analysis, Standardization, Phytochemical, Abutilon indicum, Thespesia populnea, Malvaceae.

INTRODUCTION:

In recent years, there has been a steady increase of awareness in the use of medicinal plants in developing countries because ease of its availability and safety in comparison with synthetic drugs. The medicinal importance of plants is due to the some chemical substances that produce a definite physiological action on the human body¹. The genera in the family of Malvaceae include cotton, okra, and various types of mallows. Malvaceae family consists about 243 genera and 4225 species of the herbs, shrubs, and trees in the new phylogenetic circumscription². Members of this family are prevalent in many areas of the globe and particularly in tropical and temperate regions³. Many species of Malvaceae are known by their different uses in folk medicine such as diuretic, in treatment of rheumatism, gastrointestinal disorders, snakebites, and asthma.

Anti-inflammatory and antinociceptive effects have also been investigated. Phytochemical investigations of Malvaceae species describe the isolation of flavonoids, essential oils, fatty acids, triterpenes, sesquiterpenoids, and sesquiterpene lactones ^[4].

Abutilon indicum Linn. Sweet, (Family: Malvaceae) is a hairy herb commonly known as ‘Indian mallow’. It has been a reputed remedy in the Siddha and Ayurvedic systems of medicine. Flavonoids like kaemferol, gossypetin, quercetin, essential oil, saponins, alkaloids, cyanidin glycosides, cardiac and cyanogenetic glycosides, tannins, and phenolic compounds are reported^{5, 6}.

Thespesia populnea Linn. Soland, (Family: Malvaceae) also known as Indian tulip tree has been used traditionally in various ailments⁷. Medicinal importance of this plant is mentioned in Dravyaguna. Presence of phytosterols, flavonoids, gossypetin, alkaloids, glycosides, and phenolic compounds has been reported⁸.

The main objective of the present investigation is to focus on the similarity in proximate characters and chemical constituent’s present in the two plants from the same family to aid identification of the herbs.

MATERIALS AND METHODS:

Collection and Authentication of Plant Material:

Leaves of A. indicum and T. populnea were collected from Ahemednagar District of Maharashtra. Plants were authenticated at Botanical survey of India, Pune. (Voucher No: SWITABI1, SWITTHP2)

Sample Preparation:

Powdered drug (100 gms) of both the plants were cold macerated with Methanol for 7 days. After maceration, residue was filtered and solvent was evaporated in open air. Further extract was dissolved in methanol for determination of various parameters.

Preliminary Phytochemical Screening:⁹

Preliminary phytochemical screening of MEAI and METP was carried out as per the standard procedure.

Ash Values:¹⁰

Total ash value, acid insoluble ash, water soluble ash and sulphated ash values of powdered leaves were determined by the procedure given in Indian Pharmacopoeia.

Extractive Values:¹⁰

Extractives values of powdered leaves were determined in different solvents viz. Petroleum ether, Chloroform, Ethyl acetate, Methanol, and Water.

Loss on Drying:¹⁰

Moisture content was determined by heating 2 gms of powder of leaves at 100⁰C till it achieved constant weight.

Fluorescence Powder Analysis:^11,12

Powdered leaves and extracts were treated with the different chemical reagents and color changes were observed under short and long UV light.

Total Phenolic Assay:^{13, 14}

The total phenolic content of extracts was determined by using Folin-Ciocalteu’s (FC) assay. 1 ml of extracts (50 mg/l) and standard solution of gallic acid (20, 40, 60, 80, 100 mg/l) was added to 25 ml volumetric flask, containing 9 ml of distilled deionised water. Reagent blank was prepared by omitting the samples. 1 ml of Folin-Ciocalteu’s phenol reagent was added to the mixture and shaken vigorously. 10 ml of 7 % sodium carbonate was added to the above mixture after 5 min. Volume was made upto 25 ml with distilled deionised water and mixed thoroughly. After incubation for 90 min at room temperature, the absorbance was measured at 750 nm against reagent blank. Total phenolic content of extracts was expressed as µg gallic acid equivalents/ mg extracts. Samples were analysed in duplicates using UV 1800 spectrophotometer (Shimadzu).

Total Flavonoid Assay:^{13, 14}

Total flavonoid content was measured by Aluminium Chloride assay. 1 ml of extracts (50 µl) and quercetin (20, 40, 60, 80,100 mg/l) was added to 10 ml volumetric flasks containing 4 ml of deionised water. To this flask 0.3 ml of 5 % sodium nitrate was added. After 5 min, 0.3 ml 10 % aluminium chloride was added. After this, 2 ml 1M sodium hydroxide was added and total volume was made upto 10 ml with distilled deionised water. The solutions were mixed properly and the absorbance was measured against prepared blank by omitting samples at 510 nm. Total flavonoid content of extracts was expressed as µg Quercetin equivalents / mg. Samples were analysed in duplicates by using UV 1800 spectrophotometer (Shimadzu).

Thin Layer Chromatography:¹⁵

TLC studies of methanolic extract of both the plants were carried out.

Sample preparation: 1 mg of methanolic extracts were dissolved in 10 ml Methanol

Stationary Phase: Pre-coated silica gel plates, Merck 60 F254

Chamber saturation: 30 min

Mobile phase and spraying reagents used for visualization of different phytochemicals were given in a table 5.

High Performance Thin Layer Chromatography:^{16, 17}

Chromatographic Conditions

Sample preparation: 1 mg of methanolic extracts were dissolved in 10 ml Methanol

Stationary phase: Pre-coated silica gel plates, Merck 60 F254

Mobile phase: n-Hexane: Chloroform: Ethyl Acetate: Methanol (3:2:2:3)

Lamp: Deuterium

Wavelength: 254 nm

Application mode: CAMAG Automatic TLC Sampler III

Development mode: CAMAG Twin Trough Chamber

Scanner: CAMAG TLC Scanner 3 and WinCATS software

Fourier Transform Infrared Spectroscopy:¹⁸

Powdered leaves (1 mg) were heated in oven at 50⁰C for 1 hr to remove surface moisture. It was triturated with 100 mg of KBr. The pellets were prepared by applying great pressure (upto 7 ton) to the dry mixture. Samples were analysed by using ALPHA-T IR spectroscopy (Brucker).

RESULTS AND DISCUSSION:

The concept of standardization is relatively new for Phytomedicine, but it is rapidly growing to ensure high quality products. In the field of phytomedicine, standardization applies only to extracts which contains both the active and inactive components. Standardization is required to ensure a consistent supply of high quality phytopharmaceutical products¹⁹. WHO encourages, endorse, and recommends traditional herbal remedies in National Health Care Programmes because such drugs are easily available at low cost and they are safe²⁰

Preliminary Phytochemical Screening:

It suggests the probable presence of phytoconstituents and guides for extraction. In both the plants extract (MEAI and METP) tannins, phenolic compounds and flavonoids were found in a higher amount. The details are given in Table 1.

Ash Values:²¹

Ash values are significant for drug evaluation. Ash is composed of non-volatile inorganic (Silica and metallic salts) components. It involves the oxidation of the components of drug. Sample is ignited under the specified conditions to measure the amount of the residual substances. High ash value is a measure of substitution, adulteration, contamination, and negligence in collection of crude drug. High Acid insoluble ash indicates contamination with silicious material (sand, earth). Water soluble ash indicates the amount of contents which are water soluble. Result suggests that mineral content is higher in A. indicum than T. populnea. Results are reported in Table 2.

Table 2 Ash values for A. indicum and T. populnea (%w/w)

Parameters	*A. indicum* (%w/w)*	*T. populnea* (%w/w)*
Total ash	13	10
Acid insoluble ash	1.0	0.64
Water soluble ash	5	6.1
Sulphated ash value	5	3

*Mean value of three counts

Extractive Values:

Extractive values are suggestive of amount of active constituents present in a given medicinal plant material when extracted with solvents. Presence of phyto-constituents in a particular solvent depends upon the nature of solvent used and drug. High methanol and water soluble extractive values indicate the presence of more polar constituents in both the plants. It might guide us for the isolation of maximum active constituents from plants. Results are given in Table3.

Table 3 Extractive values (%w/w) with different solvents.

Extracts	*A. Indicum* (%w/w)*	*T. populnea* (%w/w)*
Pet ether	2.1	5
Chloroform	1.5	3.2
Ethyl acetate	0.3	0.6
Methanol	8.3	8
Water	15.7	10.3

*Mean value of three counts

Loss on Drying:

If the humidity in the drug material exceeds 15 % w/w then entire drug is at risk of decaying¹⁹. The loss on drying determines both water and volatile matters in the crude drug²¹. Moisture content of A. indicum and T. populnea was found to be 10 % w/w and 7 % w/w respectively. Moisture content of the selected plants were found below 15 %w/w, hence low moisture content would hindered the growth of microorganisms and the storage life will be more.

Foreign Organic Matter:

Medicinal plants should be entirely free from stones, dust, insects, soil and any other animal contaminations²⁰. Both selected powdered plant materials were found to be free from organic matter when observed under microscope at 6X resolution.

Fluorescence Analysis:

Herbal drugs are used in a powdered form. Adulteration in the powdered form is very easy so by observing the powder under UV light it can be easily detected. Fluorescence characteristics of any powdered drug are very distinctive and helpful in distinguishing the features for the determination of a drug. Fluorescence analysis is a part of standardization which is helpful for identification of plant substitution and adulteration. The powder of leaves and crude methanolic extracts of A. indicum and T. populnea were analysed under day light (254 nm) and Ultra Violet light (365 nm). Results of analysis were given in Table 4.

Total Phenolic and Flavonoid Assay:

The term phenolic embraces a wide range of plant substances which possess in common an aromatic ring bearing one or more hydroxyl substituents. Among the natural phenolic compounds, flavonoids form the largest group. Phenolic compounds are aromatic, so that they show strong absorption in the UV and visible region of the spectrum.

Table 4 Fluorescence Analysis of A. indicum and T. populnea leaves and extracts with different chemicals.

Treatment	Visible Light		UV light
	Visible Light		Short wavelength		Long wavelength
	AI	TP	AI	TP	AI	TP
Powder	Green	Light green	Yellowish green	Green	Dark green	Dark green
Powder + 5 % KOH	Dark yellow	Dark yellow	Light green	Light green	Green yellow	yellow
Powder + 5 % NaOH	Dark yellow	Dark yellow	Light green	Bottle green	Green yellow	Dark green
Powder + 5 % FeCl₃	Yellowish brown	Yellowish brown	Dark green	Green	Bluish green	Bluish brown
Powder + I₂ solution	Brown	Brown	Brownish green	Brownish green	Dark brown	Dark green
Powder + Dil. H₂SO₄	Brown	Brown	Dark brown	Faint brown	Dark green	Blue
Powder + Conc. H₂SO₄	Brown	Brown	Blackish green	Dark green	Green	Green
Powder + Dil HCl	Brown	Brown	Greenish brown	Greenish brown	Yellow	Yellow
Powder + Conc. HCl	Brown	Brown	Green	Dark green	Yellow	Yellow
Powder + Ammonia solution	Light yellow	Brown	Yellowish green	Green	Light green	Brown
Petroleum ether Extract	Yellow	Brown	Yellow	Dark green	Dark yellow	Dark brown
Ether Extract	Yellowish brown	Light yellow	Yellowish brown	Yellowish green	Brown	Light green
Chloroform Extract	Yellowish grown	Yellow	Yellowish brown	Yellow	Dark brown	Yellow
Alcohol Extract (95%)	Green	Yellowish green	Green	Yellowish green	Brown	Yellowish green
Aqueous Extract	Brown	Orange	Green	Orange	Green	Dark red

Table 5 TLC pattern of Total methanolic extract of A. indicum and T. populnea

Constituents	Mobile Phase	Visualizing agent	Spot Color		Rf values
Constituents	Mobile Phase	Visualizing agent	AI	TP	AI	TP
Steroids	Chloroform: Benzene (5:5)	Vanillin- H₂SO₄acid	Brown	Brown	0.35	0.27
Flavonoids and Tannins	n-butanol: Acetic acid: H₂0 (4:1:5)	5% FeCl₃ solution	Blue	Blue	0.41	0.35
Triterpenoids	Benzene: Ethyl acetate:H₂0 (9.5:5)	Vanillin-H₂SO₄acid	Brown	Brown	0.85	0.46
Amino acids	96% Ethanol:H2O (7:3)	Ninhydrin solution	Pink	Pink	0.68	0.55

Spectral methods are therefore very important for the identification and quantitative analysis of phenols²². The Folin-Ciocalteu method has the advantage of a comparatively equivalent response to different phenols. TPC determination was based on the principle that oxidation of phenolic groups to phenolates due to FC reagent (a solution of phosphomolybdic and phosphotungstenic acids) in alkaline medium (7 % Na₂CO₃) giving blue color molybdenum-tungsten complex²³. Flavonoids are present in all vascular plants. Flavonoids are phenols and hence change in color is observed when treated with the base (NaOH) so that they can be easily detected in solution²². Total Phenolic content of A. indicum and T. populnea was found to be 40.20 µg/mg and 59.88 µg/mg respectively. Total flavonoid content of A. indicum and T. populnea was found to be 7.86 µg/mg and 10.54 µg/mg respectively. The results obtained from proximate analysis of leaves of the both plants establishes that they can be ranked as polyphenol rich leaves due to their relatively high phenolic content.

Thin Layer Chromatography:

Thin Layer Chromatography is a guide for the isolation and identification of constituents present in a total extract. It is a preliminary trial to select the mobile phase for HPTLC and column chromatography. By using different mobile phases and visualizing agents the some groups of compounds were identified. Results are given in Table 5.

HPTLC:²⁴

Chromatographic fingerprint suggest the possible active constituents present in the crude herbal drug. High performance thin layer chromatography (HPTLC) is a recent, powerful analytical method with the grate separation power, reproducibility and performance superior to classic TLC. HPTLC fingerprint is also suitable for rapid and simple authentication and comparison of the fine difference among samples with identical plant resource but different geographic locations and hence is a very important mean in herbal drug industry. Chromatographic fingerprinting provides information of quality control and standardisation of herbals. HPTLC fingerprint pattern of A. indicum has shown the presence of 7 spots at different Rf value indicating presence of 7 different constituents in the extract (Data shown in Fig 1 and Table 6), while methanolic extract of T. populnea has shown 9 separate peaks at different Rf values indicating presence of 9 phytoconstituents in the extract (Data shown in Fig 2 and Table 7).

Table 6 Peak display at different Rf values of MEAI

Peak	Rf	Height (AU)	Area (%)
1	0.05	12.4	0.29
2	0.48	247.5	31.39
3	0.50	253.9	42.2
4	0.70	21.1	0.87
5	0.78	22.5	1.14
6	0.88	73.1	10.43
7	0.98	93.0	13.67

Figure 1 Densitometry scan of Methanolic extract of A. indicum leaves

Table 7 Peak display at different Rf values of METP

Peak	Rf	Height (AU)	Area (%)
1	0.01	32.6	1.15
2	0.03	24.0	4.37
3	0.05	19.8	3.19
4	0.16	5.6	0.55
5	0.40	55.8	22.24
6	0.47	51.9	12.89
7	0.50	49.5	18.83
8	0.81	44.50	14.40
9	0.86	68.2	22.38

Figure 2 Densitometry scan of Methanolic extract of T. populnea leaves.

Fourier Transform Infrared Spectroscopy:

This test validates the authenticity and purity of herb samples. No two herbs have the same absorption spectra. FTIR spectrum makes the unambiguous identification of herbs²⁵. FT-IR can be simple and powerful methods for quality control of herbs. The peaks in IR spectra are due to the absorption of characteristic frequencies by molecules. Different functional groups of a molecule (like – OH, -C=O, CHO, C-H bonding and Stretching, -NH) exhibit peaks at different ranges of wavenumbers. Table 8, Fig. 3 and 4, suggest the presence of functional group in the powdered sample of both the plants. The IR absorptions displayed a hydroxyl groups at (3449 cm-¹, 3445cm-¹) , carbonyl groups at (1639 cm-¹, 1635 cm-¹) and aromatic ring at (690, 700, 750±25 cm-1and 677, 708 ) for AI and TP respectively. Presence of –OH and C=O group revealed that might be polyphenolic compounds are present in the plant which is supported by total phenolic and flavonoid contents.

Table 8 Functional group present in powdered leaves of A. indicum and T. populnea

Functional group	Frequency Range (cm^-1)
	Theoretical Values	Observed Values
	Theoretical Values	AI	TP
Alcohol, Phenols (-OH), strong, broad	3500-3300	3449	3445
Alkane (-C-H stretch), weak	2950	2956	2959
Carbonyl (C=O), strong	1610-1680	1639	1635
Alcohols, Ethers, Carboxylic acids, Esters (C-O)	1050-1300	1047, 1105, 1157, 1256, 1325	1059, 1105, 1157, 1258, 1327
Aromatic ring	690, 700, 750±25	781	677, 708

Figure 3 IR spectra of A. indicum leaves

Figure 4 IR spectra of T. populnea leaves

CONCLUSION:

The proximate analysis, physicochemical parameters, fluorescence study, preliminary phytochemical screening, HPTLC fingerprint analysis, and IR spectra can be used as an analytical tool for the correct identification of the plant. Presence of phenols and flavonoids were supported by total phenol and flavonoid content assay and also revealed the presence of –OH group in IR spectra. –C=O group is present in the compound which suggest the presence of flavonoids. By controlling the various parameters like ash values, moisture content, foreign organic matter and by determining total phenolic and flavonoid content quality plant material can be detected and used for further studies. Attempts should be made to isolate and standardise the active phytoconstituents by column chromatography. Characterisation of isolated compounds by various means like UV, IR, ¹H NMR and ¹³C NMR, and MS will be helpful. These all are the analytical tools to identified the adulterants, if any, in the crude plant material. And also by adapting all the above methods, the plants of the same genus can be differentiated.

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Received on 16.07.2011

Accepted on 03.09.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(5): Sept.- Oct.2011, 211-216

Plant	Alkaloids	Carbohydrates	Glycosides	Flavonoids	Tannins and Phenolic compounds	Proteins and Amino acids	Triterpenoid	Sterols
AI	+	+	+	++	++	+	+	-
TP	-	+	+	+++	++	+	+	+