Comparative phytochemical study of stem bark versus small branches of Acacia nilotica Linn. using HPTLC- UV detection Method

 

S. C. Verma¹*, E. Vashishth¹, S. Subhani¹, R. K. Tiwari², R. Singh¹, P. Pant¹, M. M. Padhi¹,

K. S. Dhiman¹

¹Central Council for Research in Ayurvedic Sciences,

61-65, Institutional Area, Opp.-D-Block, Janakpuri, New Delhi-110058, India

²NVRI & H, Sector-25, Indra Nagar, Lucknow

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

Acacia nilotica Linn. is a common, medium sized tree, belonging to family Leguminosae. It is popularly known as babul, kikar or Indian gum Arabic tree which has been recognized worldwide as a multipurpose tree. It has the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions and to defend against attack from predators such as insects, fungi and herbivorous mammals. Phytochemical screening of the stem bark reported that it contains terpenoids, alkaloids, saponins and glycosides which made it so useful from medicinal point of view. In this paper a new, simple HPTLC-UV pattern method is proposed for comparison of phytochemicals constituents present in stem bark and small branches of A. nilotica. The method can also be used for efficiently, identify and distinguish of different A. nilotica species. The results revealed that the phytochemical fingerprint profiling of small branches of A. nilotica was slightly similar with stem bark as an official part of A. nilotica plant, therefore small branches may not be used in place of stem bark and vice-versa. The retention factor (Rf) of A. nilotica stem bark and small branches furnished a specific HPTLC chromatogram fingerprint which might be helpful for quality assurance and detection of adulteration of crude extracts.

 

 

 

INTRODUCTION:  

The genus Acacia belongs to family Mimosaceae. Acacia Wild is a very large genus containing trees, shrubs and climbers^1. Acacia nilotica L.(Fig.1) is a common, medium sized tree, belonging to family Leguminosae². A nilotica popularly known as babul, kikar or Indian gum Arabic tree which has been recognized worldwide as a multipurpose tree^1,3. A. nilotica is possessing broad distribution from Egypt to Mauritania, South Africa in Africa and from East Asia to India, Pakistan and Iran in Asia⁴. Burma, Sri Lanka, Saudi Arabia, in West and East Sudan^{1, 5}. It is a fast growing tree in Sudan-seashore of Africa⁴.

 

 

It also grows in arid, semi-arid, hot and wet regions such as the Persian Gulf, Oman Sea, in Boushehr Province, Hormozgan Province, Sistan and Baluchestan Province (Chahbehar, Iranshahr and Nikshahre) as well as in deep loam soils⁴. In Australia it is regarded as one of the worst weeds because of its invasiveness, potential for spread, economic and environmental impacts³. In India, natural babul forests are generally found in Andhra Pradesh, Chhattisgarh, Gujarat, Haryana, Jharkhand, Karnataka, Madhya Pradesh, Maharashtra, Rajasthan, Uttar Pradesh and other dried parts of country and also widely planted in almost all states and Union territories except north-eastern states, Kashmir and Kerala^1,5.

 

According to the literature, this famous tree is of two important types. Characteristics of one tree, which is known as black consists less spines, many branches, trunk is black, wide and large. Characteristics of second tree which is known as brown have more spines than black one and its trunk is brownish black⁵.Commonly A nilotica, grows to 15-18 m in height and 23 m in diameter. In young trees the colour of bark is generally slaty green but on maturity it become nearly black with deep longitudinal fissures exposing the inner grey-pinkish slash, exuding a reddish low quality gum³. The leaves of Acacia are similar to the leaves of tamarind. They are compound, bipinnate, pinnae 3-10 pairs, 1.3- 3.8 cm long, leaflets 10-20 pairs, and 2-5mm long^3,4,5. The straight, shiny, thin, light grey spines present in auxiliary pairs, usually 3-12 pairs, 5-7.5 cm long in young trees but mature trees commonly without thorns³. Flowers are bright golden yellow coloured, round and spherical in shape⁵ with globulous heads, 1.2-1.5 cm in diameter and born either auxiliary or whorly on peduncles 2-3 cm long located at the end of branches³. The blooming season of flowers is between July to August and their pod comes in the month of February to March^5. Pods are green, 7-15 cm long and tomentose when immature whereas on maturity they become greenish black, indehiscent, deeply constricted between the seed giving a necklace appearance ³. Pods contain approx 920 seeds. In between pod and seeds, there is a thin white film. Seeds are small, compressed, ovoid, flat and broad in shape having dark brown shining colour with hard testa ³. In the beginning seeds are green and on dry they become black. Pod contains a yellowish sticky fluid. Gum exudes from the trunk or wounds in bark in the month of March to April and it is white and red in colour ^{5, 6}. Trees are tapped to increase gum yield by making incisions in the bark or treating with stress hormone ethylene or ethylene-releasing compounds such as Ethephon (2chloroethylphosphonic acid) ⁶.

 

The literature reported that Acacia species is one of the richest resources of bioactive chemical constituents such as alkaloids, flavonoids, glycoside, saponins, tannins, stearic acid, vitamin-C (ascorbic acid), carotene, phenolics, polysaccharides, terpenoids, gallic acid, phlobatannin, pyrocatechol, (+) -catechin, protocatechuic acid, (-) epi-gallocatechin-7-gallate and (-) epigallocatechin-5, 7-digallate and many other ^{2, 7, 8}. The concentration of tannin in a bark varies considerably from 12-20%. Several polyphenolic compounds have also been reported in a bark. Phytochemical screening of the stem bark reveals that it contains terpenoids, alkaloids, saponins and glycosides. The bark is also reported to contain (-) epicatechin, (+) dicatechin, quercetin, gallic acid, (+) leucocyanidin gallate, sucrose and (+) catechin-5-gallate. For the first time, in this medicinal plant the two polyphenolic compounds like kaempferol and umbelliferone have been reported. Due to its lower molecular weight of polyphenols of the bark, it is mainly responsible for fungitoxic activity⁵. The gum contains galactose, Larabinose, L-rhamnose, and four aldobiouronic acids ⁶.

Almost it’s all parts are used in medication including root, bark, leaves, flower, gum, pods etc ⁹. It is considered as a very important economic plant since early times as a source of tannins, gums, timber, fuel, fodder and medicine ⁶. A. nilotica has the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions and to defend against attack from predators such as insects, fungi and herbivorous mammals. At least 12, 000 such compounds have been isolated so far ¹⁰. Among the Acacia species, A. nilotica occupies a crucial place in the indigenous system of medicine against various diseases such as an effective medicine in the treatment of malaria, sore throat asthma, cough, flu, rheumatism, hemorrhoid, inflammation, liver infection (Hepatitis C Virus) cancers, tumors, congestion, diarrhoea, dysentery, ophthalmia, tuberculosis, leprosy, menstrual problems, plasmodial disease, bacterial disease, body vigour, wound healing, cytotoxicity, burning sensation, tonsillitis, platelets aggregation, helminthes infection, diabetes and hypolipidemia ^2,7. The roots are used against cancers and/or tumors (of ear, eye, or testicles), tuberculosis and indurations of liver and spleen ^{7, 11, 12, 13}. The bark is useful in the treatment of eczema ⁴, diarrhoea, dysentery, leprosy ⁷, colds, bronchitis, bleeding piles, leucoderma, used as antibacterial, antioxidant, anti-mutagenic, cytotoxic, acrid cooling, styptic, emollient, anthelmintic, aphrodisiac, diuretic, expectorant, emetic, nutritive, in hemorrhage, wound, ulcers, leprosy, small pox, biliousness, burning sensation, toothache, seminal weakness ^{10,12,14,15,16}. As it contains a large quantity of tannin, it is used as a powerful astringent; its decoction is largely used as a gargle and mouth wash in cancerous and syphilitic affections. Infusion of bark (1½ ounces to one pint of water) is given in chronic diarrhoea and diabetes mellitus in doses of 1½ to 2 ounces twice a day. The juice of the bark mixed with milk is dropped into the eye in conjunctivitis. Decoction of bark is largely used as an astringent douch in gonorrhoea, cystitis, vaginitis, leucorrhoea and prolapse of uterus ⁹. The leaves of Acacia are used to treat dressing of ulcers, diarrhoea, dysentery, cough, diabetes, eczema, wound healing, burning sensation and as an astringent, aphrodisiac, demulcent, anti-asthmatic etc ⁴. It is chemopreventive, anti mutagenic, anti bacterial and anticancerous ^{12, 13}. Traditionally the bark, leaves, flowers, gum and immature pod are used against cancer, cold, congestion, cough, diarrhea, dysentery, fever, gall bladder, hemorrhoid, ophthalmia, sclerosis, small pox, tuberculosis, leprosy, bleeding piles, leucoderma, and menstrual problems. These parts are also acts as anti-mutagenic, strong antioxidant, vasoconstrictor, spasmogenic, anti-asthamatic, cytotoxic, antipyretic, anti-diabetic, anti-plasmodial and antiplatelet aggregatory agents ^{8, 11}. Specially the pods are act against HIV-1 PR, Inhibited HIV-1 induced cythopathogenicity, antioxidant and shows antiplatelet aggregatory activity ^17,18,19. Seeds show spasmogenic activity and antiplasmodial activity ^{20, 21}. Gum is used as astringent, emollient, liver tonic, antipyretic and antiasthmatic ²². Its wood is also desirable for engraving and woodturning and still is used for shipbuilding and furniture. Its leaf and fruit are used as fodder. These trees are very valuable for restoring arid lands and for fixing sand dune. In the past, A. nilotica wood was cut illegally and the timber was delivered to neighbouring countries and there it was used for building large ships but later it was prevented severely by Natural Source Offices in sea shore provinces. A lot of measures have been performed for improving and restoring this national treasure ⁴.

 

Figure 1: Acacia nilotica Linn. Plant

 

Figure 2: Small Branches

 

Figure 3: Stem bark

 

MATERIALS AND METHODS:

Plant Materials and Chemicals

Plant materials i.e. small branches of stem (Fig.2) and stem barks (Fig. 3) of A. nilotica were collected in December 2013 and authenticated by Dr. R. K. Tiwari, Research Officer, Pharmacognosy, National Veterinary Research Institute & Hospital, Lucknow. All chemicals (AR grade) and TLC plates were purchased from E. Merck Pvt. Ltd. (Mumbai, India). 

 

Sample preparation

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.

 

Conventional extraction of stem bark and small branches of stem of A. nilotica were performed at room temperature (28^o ± 3^oC) 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. nilotica (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 concentrated up to 10 mL to get the sample solution of 100 mg mL^-1. 5 µL of each sample was applied separately to TLC plate for the development of fingerprints.

 

HPTLC-UV detection Method:

High Performance Thin Layer Chromatography was performed on 10 cm × 10 cm TLC plates pre-coated with 0.25 μm thin layers of silica gel 60 F₂₅₄ (E. Merck). Both samples (stem bark and small branches) 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: 8: 2:0.5 (v/v/v) and as mobile phase for both n-hexane 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 visualized under λ 254 nm and λ 366 nm for ultra violet detection and taken the fingerprints as evident in Figures 4-5.  Further, the same TLC plate was derivatized with anisaldehyde-sulphuric acid reagent and visualized in white light obtained fingerprints were as evident in Figures 6 using CAMAG Reprostar and WinCATs software (V1.4.2; CAMAG).

Table 1: R_f value of phytochemicals present in n-hexane, ethyl acetate and ethanol extract of A. nilotica (St. Bk. and Sm. Br.) at different wave-lengths.

Wave-length	n- Hexane extract		Ethyl acetate extract		Ethanol extract
	Stem bark	Small branches	Stem bark	Small  branches	Stem bark	Small branches
254	0.40, 0.84	0.40, 0.84	0.06, 0.18 0.34	0.06, 0.34	No band	No band
366	0.60, 0.68, 0.74, 0.85	0.44, 0.57, 0.60, 0.66, 0.68, 0.74, 0.85,         0.88	0.12, 0.23, 0.40	0.12, 0.23, 0.28, 0.36, 0.40, 0.51, 0.56, 0.61, 0.69, 0.75	0.08, 0.13, 0.35, 0.76, 0.80	0.35, 0.42, 0.62, 0.76, 0.80
Visible light after derivatization	0.37, 0.45, 0.52, 0.56, 0.73, 0.88	0.35, 0.37, 0.45, 0.52, 0.56, 0.82, 0.88	0.06, 0.38	0.06, 0.38, 0.51, 0.54	0.11, 0.34, 0.49, 0.60, 0.89	0.34, 0.49

 

HPTLC of ethyl acetate extract and alcoholic extract of both drugs was performed same procedure with the mobile phases of Toluene: Ethyl acetate 7:3 (v/v) and Toluene: Ethyl acetate: Formic acid 6.5:3.5:0.5 (v/v/v) respectively and then visualized in λ 254 nm, λ 366 nm and white light using CAMAG Reprostar and WinCATs software as shown in Figure 7-12.

 

 

                  1               2                                     1                 2                                  1                      2 

                     254 nm                                                     366 nm                                                 After derivatization

                         Figure 4                                                        Figure 5                                                      Figure 6    

 

 

Figure 4-6: TLC fingerprint of n- hexane extract of A. nilotica (1= St. Bk.; 2= Sm. Br.) 

 

             1                   2                                      1                      2                                  1                 2 

                      254 nm                                                      366 nm                                           After derivatization

                             Figure 7                                                        Figure 8                                                     Figures 9                            

Figure 7-9: TLC fingerprint of ethyl acetate extract of A. nilotica (1= St. Bk.; 2= Sm. Br.)

 

                  1               2                                     1                 2                                      1             2 

                      254 nm                                                      366 nm                                               After derivatization

                          Figure 10                                                          Figure 11                                                     Figures 12      

Figure 10-12: TLC fingerprint of ethanol extract of A. nilotica (1= St. Bk.; 2= Sm. Br.)

 

RESULTS AND DISCUSSION:

No such study was found in literature for comparative phytochemical study of stem bark versus small branches of A. nilotica Linn by using High Performance Thin Layer Chromatographic-Ultra Violet detection Method. Comparative study of TLC fingerprints of stem bark and small branches of A. nilotica revealed that many similarities in phytochemical fingerprints were found and evident in Table-1 and Fig. 4-12.

 

Phytochemical fingerprints of n-hexane extract of stem bark and small branches showed two common bands at R_f 0.40 (black) and 0.84 (blue) under UV detection at 254 nm. Under 366 nm UV detection, stem bark and small branches showed four and eight bands respectively, out of which three bands were found similar at R_f  0.60 (red)  0.74 (red) and 0.85 (blue). After TLC plate derivatized with Anisaldehyde sulphuric acid reagent and visualized under white light, stem bark and small branches showed six and eight bands respectively, out of which five bands were common at R_f 0.37 (brown), 0.43 (blue), 0.52(violet), 0.56 (purple) and 0.88 (blue ) as represented in Table 1 and Fig. 4-6.

 

Phytochemical fingerprints of ethyl acetate extract of stem bark and small branches showed three and two bands respectively, out of which two bands were found similar at R_f 0.40 (black) and 0.84 (blue) under UV detection at 254 nm. Under 366 nm UV detection, stem bark and small branches showed three and ten bands respectively, out of which three bands at R_f 0.12 (blue), 0.23 (light blue) and 0.40 (blue) were found similar. After derivatized with Anisaldehyde sulphuric acid reagent and visualized under white light, stem bark and small branches showed two and four bands respectively, out of which two bands were found similar at R_f 0.06 (brown) and 0.38 (blue) as showed in Table 1 and  Fig. 5-8.

 

Phytochemical fingerprints of ethanol extract of stem bark and small branches under UV detection at 254 nm, showed no band in both parts. While under 366 nm UV detection, stem bark and small branches both showed five bands, out of which three bands at R_f 0.35 (red), 0.76 (red) and 0.80 (red) were found similar. After TLC plate derivatized with Anisaldehyde sulphuric acid reagent and visualized under white light, stem bark and small branches showed five and two bands respectively, out of which two bands at R_f 0.34 (blue) and 0.49 ( blue) were found similar in both parts (St. Bk. and Sm. Br.) as evident in Table 1 and Fig.10-12.

 

CONCLUSION:

The phytochemical fingerprint profiling of small branches of A. nilotica were slightly similar with stem bark as a official part of A. nilotica plant, therefore small branches may not be used in place of stem bark and vice-versa. The R_f helped in evaluation of phytochemical diversity in different parts of A.nilotica. TLC phytochemical fingerprint profiling of n-hexane, ethyl acetate, ethanolic extracts of stem bark and small branches of A. nilotica have been given an idea about the presence of various phytochemicals in their reported parts. The TLC spots provided valuable clue regarding presence or absence of various phytochemicals or metabolites of the plants.

 

ACKNOWLEDGMENTS:

Authors are thankful to Director General, Central Council for Research in Ayurvedic Sciences, New Delhi to provide the financial support under IMR scheme -2^nd PEMC 2013 for this research work.

 

CONFLICT OF INTEREST:

Authors have no conflict of interest.

 

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Received on 26.03.2015         Modified on 08.04.2015

Accepted on 12.04.2015         © AJRC All right reserved