Simple, Rapid and Precise Method for Quality Assessment of Different Parts of Aegle marmelos L. used in Indian System of Medicine

 

S. C. Verma1*, R. Rani1, E. Vashishth1, K. Basu2, P. Pant1 and M. M. Padhi1

1Central Council for Research in Ayurvedic Sciences, 61-65, Institutional Area, Opp.-D-Block, Janakpuri, New Delhi-110058, India

2Arbro Pharmaceuticals (Pvt.) Limited, Kirti Nagar, New Delhi, India

 

ABSTRACT:

Aegle marmelos (L.) Corr. Serr, family Rutaceae is an important medicinal plant and immensely used in the Indian System of Medicine to cure human diseases. Plant parts like root, stem bark and fruit have been reported for various medicinal properties such as anti-diabetic, anticancer, anti bacterial, anti fungal, anti pyretic, analgesic, antioxidant, cardio protective, radio protective, anti-diarrheal, anti-dysentery, anti-ulcer, wound healing and many more. A. marmelos is commonly known as a bael in India. Chemo-profiling screening on different parts of A. marmelos plants revealed variations in phytochemicals within different parts of plant. The unique properties of the chromatographic fingerprint were validated by analyzing root, stem bark and fruit of A. marmelos. Our results revealed that the chromatographic fingerprint combined with similarity measurement could efficiently identify and distinguish A. marmelos from the other investigated Aegle species. In this paper a new, simple method is proposed in which the TLC pattern of the extracts of root, stem bark and fruit of A. marmelos content is used for effective and reliable quality control of the drug. The method can also be used for identification of different A. marmelos species. The proposed method uses cold- extraction then clean-up by solid-phase extraction before chromatographic analysis. The results revealed that the retention factor (Rf) of A. marmelos stem bark, root and fruit furnishes a specific TLC chromatogram fingerprint which might be helpful for quality assurance and detection of adulteration of crude extracts. The root, stem bark and fruit of A. marmelos L. were also physico-chemically standardized as per WHO specification.

 

KEY WORDS: Aegle marmelos L., chemo-profiling, TLC–UV detection, physicochemical analysis

 

1. INTRODUCTION:

Aegle marmelos (L.) is a native plant of India. A. marmelos belongs to Rutaceae family and commonly known as wood apple, bilwa or bael1-3. In India, A. marmelos is grown as a temple garden plant and the leaves are used to pray Lord Shiva. A. marmelos is a subtropical plant and grows up to an altitude of 1,200 m altitude from sea level. It grows well in the dry forests on hilly and plain areas. A. marmelos is a widely distributed plant and found in India, Ceylon, China, Nepal, Sri Lanka, Myanmar, Pakistan, Bangladesh, Nepal, Vietnam, Laos, Cambodia, Thailand, Indonesia, Malaysia, Tibet, Sri Lanka, Java, Philippines and Fiji4.

 

The bael is a holy plant and its all parts are very useful, generally it is seen that if one part of any plant show any pharmacological effect then there is a major possibility that the other part give the same or related activity.

Fruit is useful in  dysentery, diarrhea, gastric troubles, constipation, laxative, tonic, digestive, stomachic, brain and heart tonic, ulcer, antiviral activity while root bark shows action against intermittent fever and fish poison, palpitation, melancholia, anti dog bite, gastric troubles, heart disorders, fever, antiamoebic, hypoglycemic, rheumatism5and stem bark are used as antipyretic6. Various therapeutic activities of the plants prompted us to further develop a simple, rapid and precise chemo-profiling method for quality evaluation of different parts of A. marmelos.

 

Bael is reported to have number of phytoconstituents like coumarins, alkaloids, steroids, and essential oils. Root and fruits contain coumarins such as scoparone, scopoletin, umbellliferone, marmesin and skimming. Fruits in addition contain xanthotoxol, imperatorin and alloimperatorin and alkaloids like aegeline and marmelline. It also contains polysaccharides like galactose, arabinose, uronic acid and L-rahaminose, which may obtain after hydrolysis. Different types of carotenoids have been reported in the A. marmelos, these are responsible for the imparting yellow pale colour to fruit. Marmelosin, skimmianine and umbelliferone are the therapeutically active principale of bael plant. Minor constituents are like ascorbic acid, sitosterol, crude fibers, tannins, α-amyrin, carotenoids, and crude proteins are also present. Apart from these chemical constituents more than 100 compounds have been isolated these are skimminine, aegelin, lupeol, cineole, citral, citronellal, cuminaldehyde, eugenol, marmesinin, marmelosine, luvangetin, aurapten, psoralen, marmelide, fagarine, marmin, and tannins have been proved to be biologically active against various major and minor disease7-17. Different studies have shown that nutritional value of bael fruit has significant mineral and vitamin contents. It also contain moisture 64.2%, protein 1.8%, fat 0.2%, mineral 1.5%, fibre 2.2%, carbohydrate 31.8%, calcium 0.06%, phosphorous 0.05%, potassium 0.6%, vitamin C 0.01%, riboflavin 1.2%, nicotinic acid 0.9%, thiamin 0.01% and iron 0.3% per 100 gm18-19. The selection should be mainly based on the ethano-pharmacological selection process that is based on the therapeutic use of plant species by a specific ethnic group20.

Herbal drugs have been increasingly appreciated as effective remedies in many countries21. For better development of herbal drugs for effective therapy, it is imperative to control the quality of the herbal drugs. Fingerprint technology has recently been introduced and accepted by the WHO and the State Food and Drug Administration (SFDA) of China as a strategy for evaluation of the quality of herbal drugs and their products22. Among these fingerprinting techniques, chromatographic fingerprinting is a very useful and popular analytical approach because it emphasizes the systemic characterization of sample composition23. Chromatographic methods currently available for fingerprinting include high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC)24-26. Many approaches have recently been developed for the qualitative and quantitative analysis of the major constituents in A. marmelos; among these, HPLC and TLC are most frequently reviewed elsewhere27–28. Compared with TLC, HPLC has the advantages of high analytical efficiency and precision. To optimize the conditions used to obtain the TLC fingerprint, the effect of solvent, drug particle size, extraction method, analysis time, and elution conditions were scrutinized. Nonetheless, the problem of whether the established TLC fingerprint could be used for effective evaluation of the quality of A. marmelos or whether this method was superior to others for assessment of the species differences remained largely unsolved. In this study, we combined chemometric methods, for example, similarity evaluation and physicochemical analysis, with TLC–UV detection to develop a specific and valid chromatographic fingerprinting approaching for quality assessment and plant part differentiation of A. marmelos.

 

2. EXPERIMENTAL:

2.1 Chemicals and Plant Materials:

All chemicals (AR grade) and TLC plates used were purchased from E. Merck Pvt. Ltd. (Mumbai, India).  Authentic plant parts (root, stem bark and fruit) of A. marmelos were collected from survey units of Central Council for Research in Ayurvedic Sciences, New Delhi, India. The plant parts were dried under a gentle stream of air in the laboratory till no loss in weight (temperature 30± 20C and relative humidity 50 ± 5%) and powdered in an electric grinder.

 

2.2 Sample preparation:

Conventional extraction of root, stem bark and fruit of A. marmelos were performed at room temperature (28o ± 3oC) with a variety of solvents ranging from non-polar to polar ones, i.e. n-hexane, ethyl acetate and ethanol. Dried and powdered parts of A. marmelos (10 g each) were extracted three times (3 × 50 mL) for 18 h of each extraction with each of the above-mentioned solvents separately. Each extract was filtered by using Whatman filter paper no. 1 and the solvents were removed under vacuum at 50°C, separately and lypholized till each extract was free from solvents. Each dried extract of root, stem bark and fruit (1 g each) of A. marmelos was dissolved separately, in 10 mL of each solvent n- hexane, ethyl acetate and alcohol, respectively to get the sample solution of 100 mg mL-1, 4 mL of each stack sample solution was taken in a 100 mL volumetric flask separately and added corresponding solvent in to each flask up to mark, to get the concentration of each sample solution is 4 mg mL-1. Each sample solutions were filtered through 0.45 µm membrane filters and 5 µL of each sample was applied separately to TLC plate for the development of fingerprints.

 

2.3 Chromatography:

Chromatography was performed on 10 cm × 10 cm TLC plates precoated with 0.25 μm thin layers of silica gel 60 F254 (E. Merck). Samples were applied on the plates as bands 10 mm wide by use of a Linomat-IV applicator (CAMAG, Switzerland) fitted with a 100 μL syringe (Hamilton, Switzerland). The application positions X and Y were both 10 mm, to avoid edge effects. Linear ascending development to a distance of 80 mm with Toluene : Ethyl acetate : Acetic acid : 9.0 : 1.0 : 0.1, (v/v/v) as mobile phase for both n-hexane extract and ethyl acetate extract was performed in a twin-trough glass chamber (20 cm × 10 cm) previously saturated with vapours of mobile phase for 20 min. The plates were dried in air and photo documented at λ 254 nm and λ 366 nm (Figures 1 - 2). Further, the plate was derivatized with anisaldehyde-sulphuric acid reagent and visualized in white light (Figures 1 - 2) using CAMAG Reprostar and WinCATs software (V 1.4.2; CAMAG). TLC of alcoholic extract was performed same procedure with the mobile phase Toluene : Ethylacetate : 7.5 : 2.5, (v/v) and visualized in λ 254 nm, λ 366 nm and white light using CAMAG Reprostar and WinCATs software as shown in Figure 3.

                                       

Figure 1(A-C): HPTLC fingerprint of n-hexane extract of three different parts (R= root; S= stem bark; F= fruit) at 254 nm (1A); 366 nm (1B); after derivatized with Anisaldehyde – sulphuric acid reagent (1C)

 

Figure 2(A-C): HPTLC fingerprint of ethyl acetate extract of three different parts (R= root; S= stem bark; F= fruit) at 254 nm (2A); 366 nm (2B); after derivatized with Anisaldehyde – sulphuric acid reagent (2C)

 

Figure 3 (A-C): HPTLC fingerprint of ethanol extract of three different parts (R= root; S= stem bark; F= fruit) at 254 nm (3A); 366 nm (3B); after derivatized with Anisaldehyde – sulphuric acid reagent (3C)

2.4 Physicochemical analysis of root, stem bark and fruit of A. marmelos:

2.4.1 Foreign Matter:

4 g of the sample was taken in a thin layer on dish. Spread the sample on dish and examined in daylight with unaided eye. If there is any suspected particle then transferred to a petridish.  Examine with 10 × lens in daylight and weigh. Record the value and calculate the percentage with respect of sample taken29.

 

2.4.2 Total ash:

2g of the sample was taken accurately in a previously ignited and tarred Silica dish. Spread the material evenly and ignite in a muffle furnace by gradually increasing the temperature to 6000C until it is white, indicating the absence of carbon. Cool the dish in desiccators and weigh. If carbon free ash cannot be obtained in this manner, cool the dish and moisten the residue with about 2 ml of water or a saturated solution of Ammonium nitrate. Dry on a water-bath, and then ignite in the muffle furnace to constant weight. Cool the dish in desiccators further 30 minutes, and then weigh. Calculate the percentage of total ash of air dried material29.

 

2.4.3 Acid insoluble ash:

To the dish containing the total ash, add 25 ml of hydrochloric acid and water (1:5) cover with a watch glass and boil gently for 5 minutes. Rinse the watch glass with 5 ml of hot water and add the washings to the dish. Collect the insoluble matter on an ash less filter paper (Whatmann No. 41) and wash with hot water until the residue is free from acid. Transfer the filter paper containing the insoluble matter to the original dish, dry and ignite to constant weight. Cool the dish in desiccators for 30 minutes, and then weigh. Calculate the percentage of acid insoluble ash of the air-dried material29.

 

2.4.4 Water-soluble extractive:

4 g of the sample was taken in a glass stopper flask. Add 100 ml of distilled water, with shake occasionally for 6 hours and then allow standing for 18 hours. Filter the solution and pipette out 25 ml of the filtrate in a pre-weighed 100 mL beaker and evaporate to dryness on a water bath. Keep it in an air oven at 105 °C for 6 hours, cool in desiccators for 30 minutes and weigh. Repeat the experiment twice, and take the average value29.

 

2.4.5 Alcohol-soluble extractive:

4 g of the sample was taken in a glass stopper flask. Add 100 mL of distilled alcohol shake occasionally for 6 hours and then allow standing for 18 hours. Filter the solution and pipette out 25 ml of the filtrate in a pre-weighed 100 mL beaker and evaporate to dryness on a water bath. Keep it in an air oven at 105°C for 6 hours, cool in desiccators for 30 minutes and weigh. Calculate the percentage of Alcohol extractable matter of the sample. Repeat the experiment twice, and take the average value29.

 

3. RESULT AND DISCUSSION:

TLC Comparative study of root, stem bark and fruit of Aegle marmelos L. reveled that many similarities in phytochemical fingerprints were found and evident in Figures 1-3. n-hexane extract of root and fruit showed under UV-254 nm two bands are similar at Rf  0.14, 0.42 (both grey). While, n-hexane extract of root showed six bands under UV-366 nm at Rf  0.10, 0.14, 0.22, 0.30 (all dark blue), 0.42 (blue) and 0.54 (dark blue) similarly stem bark showed five bands except at Rf 0.42 (blue) and fruit showed four bands except at Rf 0.10, 0.30 (dark blue). After derivatization under white light n-hexane extract of root showed three bands at Rf  0.22, 0.28, 0.46 (all violet) similarly stem bark showed two bands at Rf  0.22, 0.46 (both violet) and  fruit showed two bands  at Rf  0.22, 0.28 (both violet) and Rf data of Figures 1A-C are complied in Table 1. The TLC of ethyl acetate extract of stem bark and fruit (Figure 2A) showed under UV- 254 nm one bands at Rf  0.20 (grey) is common while no similarity was found with root extract.  Ethyl acetate extract of roots (Figure 2B) showed four bands under 366 nm at Rf 0.10, 0.12, 0.16, 0.20 (all dark blue) while stem bark extract showed six bands at Rf 0.10, 0.12, 0.16, 0.20, 0.28, 0.33 (all dark blue) similarly fruit extract showed two bands at Rf 0.20 (dark blue), 0.28 (blue) are common. After derivatization under white light (Figure 2C) the ethyl acetate extracts of root and stem bark showed five bands similar at Rf 0.20, 0.28, 0.33, 0.46 (all grey), 0.58 (violet) while fruit extract showed two bands at Rf 0.33, 0.46 (both grey) were found common in Table 2. 

 

The TLC of ethanol extract of root (Figure 3A) showed under  254 nm three bands at Rf  0.07,  0.22, 0.66 (all grey) similarly stem bark showed two bands at Rf  0.07,  0.22 (both grey) and fruit extract showed two bands at Rf  0.22, 0.66 (both grey)] are common with root. TLC profile of ethanol extract of root and stem bark (Figure 3B) showed ten bands under 366 nm at Rf 0.07, 0.12, 0.22, 0.30, 0.35 (all dark blue), 0.40 (blue), 0.46, 0.58 (both dark blue), 0.68 (blue), 0.76 (dark blue) similarly ethanolic fruit extract (Figure 3B) showed six bands under 366 nm at Rf 0.07, 0.12 (both blue), 0.22 (greenish-blue), 0.30, 0.46 (both blue), 0.68 (greenish-blue)] are common.  After derivatization under white light (Figure 3C) ethanolic root extract showed four bands at Rf 0.07, 0.46, 0.68, 0.76 (all violet) while stem bark extract showed three bands at Rf 0.07, 0.46, 0.68 (all violet) are similar with root, similarly fruit extract showed three bands at Rf 0.46, 0.68, 0.76 (all violet) are common with root extract (Table 3). 

 

The root, stem bark and fruit of A. marmelos L. were physico-chemically standardized in term of determination of extractive value and ash value. Powder of root, stem bark and fruit showed 5.68%, 9.68% and 10.12% ash content respectively. Acid-insoluble ash content for root, stem bark and fruit showed 0.86%, 0.74% and 0.72% respectively which is found under the limit as revealed by WHO. There is negligible amount of foreign matter and less amount of siliceous matter was present in the different parts of plant. Alcohol soluble extractive was found in root, stem bark and fruit showed 7.46%, 7.33% and 7.42% respectively indicated the presence of polar constituents and non-polar secondary metabolites present in the plant while water-soluble extractive was found in root, stem bark and fruit showed 14.17%, 15.17% and 13.42% respectively indicated the presence of sugar, acids and inorganic compounds. The percentage of total ash, acid-insoluble ash, alcohol-soluble extractive, water soluble extractive and content of heavy/toxic metal were found under the permissible limit of WHO29, results are depicted in Table 4.

 

 

 

Table 1: Rf value of spots present in n-hexane extract of root, stem bark and fruit of A. marmelos

Plant part

254 nm (Rf)

 

366 nm  (Rf)

 

After anisaldehyde sulphuric acid reagent (Rf)

Root

0.10, 0.14, 0.22, 0.27, 0.42, 0.54 (all grey)

0.10, 0.14, 0.19, 0.22, 0.30 (all dark blue); 0.35, 0.42, 0.50 (all blue); 0.54 (dark blue)

0.14, 0.22, 0.28, 0.35, 0.46,  0.28, 0.54 (all violet)

Stem bark

0.79, 0.98 (all grey)

0.10, 0.14, 0.22, 0.30 (all blue); 0.39 (green); 0.54 (blue)

0.22, 0.32, 0.46 (all violet)

Fruit

0.14, 0.42 (all grey)

0.14 (dark blue); 0.22(blue); 0.25, 0.28, 0.42 (all green); 0.54 (dark blue)

0.22, 0.28, 0.35, 0.42, 0.76, 0.94 (all violet)

 

 

Table 2: Rf value of spots present in ethyl acetate extract of root, stem bark and fruit of A. marmelos

Plant part

254 nm (Rf)

 

366 nm (Rf)

 

After anisaldehyde sulphuric acid reagent (Rf)

Root

0.10, 0.22, 0.25, 0.28, 0.33, 0.58 (all dark blue)

0.10, 0.12, 0.16, 0.20, 0.25, 0.28, 0.33, 0.58 (all dark blue)

0.12, 0.16, 0.20, 0.28, 0.33, 0.46 (all grey); 0.58 (violet); 0.61 (grey)

Stem bark

0.20 (grey)

0.10, 0.12, 0.16, 0.20, 0.28, 0.33(all dark blue); 0.44 (light green); 0.58 (blue)

0.20, 0.28, 0.33, 0.46 (all grey); 0.58 (violet)

Fruit

0.20, 0.45 (both grey)

0.20 (dark blue); 0.28 (blue); 0.33, 0.47 (green)

0.33, 0.46, 0.76, 0.85 (all grey)

Table 3: Rf value of spots present in ethanol extract of root, stem bark and fruit of A. marmelos

Plant part

254 nm (Rf)

 

366 nm (Rf)

 

After anisaldehyde sulphuric acid reagent (Rf)

Root

0.07, 0.22, 0.33, 0.50 (all blue); 0.66 (light grey); 0.78 (light blue)

0.07, 0.12, 0.22, 0.30, 0.35, (all dark blue); 0.40 (blue); 0.46, 0.58 (both dark blue); 0.68 (blue); 0.76 (dark blue)

0.07, 0.12, 0.22 (all violet); 0.30 (light grey); 0.35, (light blue); 0.46, 0.58, 0.68, 0.76 (all violet)

Stem bark

0.07, 0.22 (both blue)

0.07, 0.12 (both dark blue); 0.22 (blue); 0.30 (dark blue); 0.35, 0.40 (both blue); 0.46, 0.58 (both dark blue); 0.68 (greenish-blue); 0.76 (dark blue); 0.85 (light red)

0.07, 0.46, 0.68 (all violet)

Fruit

0.22 (light blue); 0.66 (grey)

0.07, 0.12, (both blue); 0.22 (greenish-blue); 0.30, 0.46 (both blue);  0.68 (greenish-blue)

0.46, 0.68, 0.76 (all violet)

 

 

Table 4: Physico-chemical parameters of Aegle marmelos (root, stem bark and fruit)

S.No.

Parameters

Root

Stem

bark

Fruit

1.         

Foreign matter

Nil

Nil

Nil

2.         

Ash content

(% w/w)

5.68

9.68

10.12

3.         

Acid Insoluble ash (% w/w)

0.86

0.74

0.72

4.         

Alcohol soluble extractive value (% w/w)

7.46

7.33

7.42

5.         

Water soluble extractive value

(% w/w)

14.17

15.17

13.42

6.         

Heavy/toxic metals in ppm by ICP-MS

Lead (Pb)

≤ 1.477

≤ 0.990 

≤ 0.248 

Cadmium (Cd)

≤ 0.113

≤ 0.085 

< 0.001  

Arsenic  (As)

≤ 0.066

≤ 0.140 

< 0.001 

Mercury (Hg)

<0.001 

< 0.001  

< 0.001  

 

4. CONCLUSION:

Chemical profiling of various parts of A. marmelos indicated that different types of phytoconstituents present in each part but many similarities in fingerprinting are found in root and stem bark. The chemical profiling of fruit is differed from root and stem bark, therefore fruit may not be used in place of root or stem and vice-versa. The Rf helped in evaluation of phytochemical diversity in different parts of A. marmelos. The phytochemical diversity was found more in stem bark followed by root and fruit at one geographical region. TLC profiling of ethanolic extracts of root, stem bark and fruit of A. marmelos has been given an idea about the presence of various phytochemicals in their reported parts. Differences in Rf value of various phytochemicals provide valuable clue regarding their polarity and selection of solvents for separation of phytochemicals which will be helped in assessment of quality control. The levels of heavy/toxic metals in A. marmelos were found in permissible limits.

 

5. ACKNOWLEDGMENTS:

This research was supported by Director General, Central Council for Research in Ayurvedic Sciences, New Delhi. Ms. Ektaa Vashishth, Senior Research Fellow (Chemistry) is highly appreciated for her assistance.

 

 

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Received on 01.08.2013

Modified on 05.09.2013

Accepted on 12.09.2013

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 5(5): September – October 2013, 236-243