Quality Evaluation of Ayurvedic Crude Drug Daruharidra, Its Allied Species, and Commercial Samples from Herbal Drug Markets of India

Berberis aristata known as “Daruharidra” in Ayurveda is a versatile medicinal plant used singly or in combination with other medicinal plants for treating a variety of ailments like jaundice, enlargement of spleen, leprosy, rheumatism, fever, morning/evening sickness, snakebite, and so forth. A major bioactive marker of this genus is an alkaloid berberine, which is known for its activity against cholera, acute diarrhea, amoebiasis, and latent malaria and for the treatment of oriental sore caused by Leishmania tropica. Although the roots of B. aristata are considered as the official drug (Ayurvedic Pharmacopoeia of India), the study revealed that different species of Berberis, namely. B. asiatica, B. chitria, and B. lycium are also used under the name of Daruharidra in different parts of the country. Detailed physicochemical and phytochemical studies of subjects like total ash, acid insoluble ash, tannins, and total alkaloids were calculated from the shade dried powdered material according to the recommended procedures. Further, heavy metal studies and quantitative estimation of berberine through HPTLC have also been performed as per ICH guidelines. A detailed study of four Berberis species, namely B. aristata, B. asiatica, B. chitria, and B. lycium, which are implicated as Daruharidra and collected from wild and ten commercial samples procured from various important drug markets in India has been carried out, which may be useful to pharmaceutical industries for the authentication of the commercial samples and exploring the possibilities of using other species as a substitute of B. aristata.


Introduction
Berberis aristata known as "Daruharidra" in Ayurveda is a versatile medicinal plant used singly or in combination with other medicinal plants for treating a variety of ailments like jaundice, enlargement of spleen, leprosy, rheumatism, fever, morning/evening sickness, and snakebite, and so forth [1][2][3][4]. In addition, the decoction of root or stem of Berberis known as "Rasaut" is speci�cally used in eye disease, skin disorders, and indolent ulcers. Its use in the management of infected wounds has also been described in Ayurvedic classical texts [5]. e major alkaloid of the plant is berberine, which is known for its activity against cholera [6], acute diarrhea [7], amoebiasis, and latent malaria and for the treatment of oriental sore caused by Leishmania tropica [4].
Although the roots of B. aristata are considered as the official drug [8], the study revealed that different species of Berberis, namely, B. asiatica, B. chitria and B. lycium are also used as Daruharidra in different parts of the country. In southern India, however, Coscinium fenestratum is used as "Daruharidra." e study also shown that most of the market material sold as Daruharidra consists of mostly the stem parts than the roots of Berberis species.
As such there are different alkaloids available to differentiate different Berberis species. Several workers have also done molecular analysis of different Berberis species including the  presented four species which re�ects the use of molecular markers and se�uence analysis for identi�cation at inter-and intra-speci�c level �9-12].
Over exploitation of B. aristata created scarcity of the material that opened new vistas to identify a possible substitute for this species. During the market surveillance of different herbal drug markets of India, it was observed that almost all the markets either comprise Berberis lycium or Berberis asiatica. Although a detailed pharmacognostic study of B. aristata, B. asiatica, and B. chitria is reported by Srivastava et al. [13][14][15], market surveillance is not yet performed. Hence, the present study has been undertaken, which may be useful to pharmaceutical industries for the authentication of the commercial samples and to explore the possibilities of using other species as a substitute of B. aristata.

Materials and Methods
e plant materials were collected from the Dhanaulti (Uttaranchal) region of India (LWG 221238-11) and the roots were preserved in 70% ethyl alcohol for histological studies. Procurement of commercial samples was done from various important drug markets of India, namely, Aligarh, Amritsar, Bangalore-I, Bangalore-II, Delhi, Hyderabad, Jammu, Lucknow, Trichur, Varanasi, and so forth. Microtome sections were cut and stained with safranin and fast green and photographed with Nikon F70X camera [16]. Physicochemical and phytochemical studies like total ash, acid insoluble ash, tannins, and total alkaloids were calculated from the shade dried powdered material according to the recommended procedures [17][18][19]. Further, heavy metal studies and quantitative estimation of berberine through HPTLC have also been performed as per ICH guidelines.

Results and Discussion
Morphological studies showed certain minor variations in all the four Berberis species (see Table 4). For example, in B. aristata and B. chitria the cut surface is bright yellow while that of B. asiatica and B. lycium is lemon yellow, and deep yellow, respectively. Similarly the colour of wood bark has also minor variation, namely, it is yellowish brown to yellowish gray in all the three species except in B. lycium the colour is grayish white. Likewise the numbers of pericyclic �bres are di�erent in all the four species, for example, the maximum is found in B. aristata and minimum in B. lycium (Tables 1, 2, and 3; Figures 1, 2, 3, and 4).
A comparative account of all physicochemical values has been depicted in histograms (Figures 5-10). It is quite clear from these studies that no signi�cant variation was observed in total ash of all the four species of Berberis. However, the percentage of acid insoluble ash of roots and stem showed signi�cant variations; for example, the highest percentage of 0.26% acid insoluble ash was observed in B. asiatica root and the lowest one of 0.05% was noted in B. aristata root ( Figure  5). It is interesting to note that the percentage of alcohol and water-soluble extractives were higher in root as compared to stem except in B. chitria ( Figure 6). On the contrary the percentage of starch was higher in stem (14-19%) except in B. lycium (root) it was 26.03%. Percentage of tannin was more or less similar in both in root and stem of all the samples (0.7-1.7%).
Similarly, the percentage of successive Soxhlet extractive values revealed that alcohol, acetone, and water extractives were found to be signi�cantly higher in B. aristata root, that is, 7.83%, 6.51% and 5.96% respectively. While the roots  of B. asiatica possessed maximum percentage of alcoholic, acetone, and water-soluble matter, that is, 10.30%, 5.92%, and 4.92%, respectively. On the other hand the percentage of successive extractives in stem was maximum in B. lycium that is, 5.11% (acetone), 7.20% (alcohol), and 3.20% (water), respectively, and percentage of hexane soluble matter was higher in B. aristata (Figure 9). e percentage of total crude alkaloid percentage was also estimated and it was found that it varied from species to species, that is, maximum in roots of B. chitria (3.65%) followed by the roots of B. lycium (2.8%), B. aristata (2.45%), and B. asiatica (2.4%), respectively. Besides, the active constituents berberine one of the major alkaloids was also calculated through HPTLC densitometric method (solvent system, n-propanol: water: formic acid, 90 : 80 : 0.4) and it was found more in roots as compared to stem, that is, 2.25-5.20% and 1.02-2.01%, respectively. Its concentration was also varied from species to species, that is, maximum in roots of B. chitria (5.20%) followed by B. lycium (3.99%), B. aristata (3.55%), and B. asiatica (2.25%). Details are depicted in Figure 10.
A comparative study of official drug B. aristata sample with that of commercial samples was made (Figures 5-15) and it was found that the Bangalore-I sample has all the similar morphological characters of roots of B. asiatica, namely, (i) outer surface grayish brown with 2 mm thick friable bark, which was separated out immediately leaving muddy yellow surface of the wood; (ii) transversely cut surface lemon yellow; (iii) sclerieds mostly in groups of 2-12 rarely solitary and comparatively more than other three  Furthermore, the commercial samples of Delhi and Lucknow showed close resemblance with the stem of B. asiatica by the presence of (i) �ne longitudinal ridges and small warts on the outer surface of bark and yellowish creamy transverse cut surface; (ii) dark brown outer surface of wood as appeared aer peeling off the bark; (iii) sclerieds rarely solitary mostly in groups of 2-12 and comparatively more than other three species; (iv) pericycle �bres interrupted by stone cells; (v) trachieds up to 680 (±167.0) and vessels up to 600 (±102.0) long (vi) pith.
Similarly, the market samples of Amritsar and Jammu were found to be the mixture of stem and root of two different Berberis species. Amritsar samples were found to be the stem  On the other hand some of the pieces of this sample showed close resemblance with that of the stem of B. asiatica in having (i) outer surface grayish brown with 2 mm thick 0 friable bark which was separated out immediately leaving muddy yellow surface of the wood; (ii) transverse cut surface lemon yellow (iii) sclerieds rarely solitary mostly in groups of 2-12 and comparatively more than other three species (iv) pericycle �bres interrupted by stone cells; (v) length of the e percentage of successive extractives (Soxhlet), sugar, starch, and berberine content also support the above �nding (Figures 6-10).
On the contrary the Hyderabad commercial sample seems to be the mixture of three Berberis species. Some pieces showed close affinity with roots of B. asiatica; other pieces resembled roots of B. aristata. Besides some of the pieces have no resemblance with any of the four species studied. ese may be the roots of B. tinctoria as it is a common species found in these areas. e samples procured from Trichur and Bangalore II do not resemble any of the Berberis species studied. Presence of wedge shaped medullary rays and wheel shaped transverse section indicate that these pieces may belong to Menispermaceae. Hence an attempt was made to compare the characters of these samples with the root and stem of Coscinium fenestratum. Aer comparison it was found that samples procured from Trichur and Bangalore II were root and stem of C. fenestratum Gaertn, respectively.
From the heavy metal analysis of crude drug samples of different herbal market, it was observed that the concentration of majority of heavy metals, namely, Cd, Co, Mn, Zn, and Cu within the permissible limits of WHO in both the collected and commercial samples. However, the signi�cant variations in Lead concentration were observed in both samples that is, in collected as well as in commercial ones. For example, in the stem of collected samples, the increase is quite low but in some of the commercial samples, namely, Amritsar, Delhi, and Hyderabad many fold increase in lead concentration was observed, this may be due to vehicular pollution or may be due to edaphic factors ( Figure 11).

Conclusion
From the ongoing discussion it is quite clear that most of the commercial samples consist of mixture of roots or stems of Berberis asiatica while B. aristata is found only in the market samples of Amritsar and Hyderabad mixed with roots of B. asiatica.
On the basis of the above study it may be concluded that in India different Berberis species and their parts are being sold in the name of crude drug Daruharidra (Table 5).