Integrating macro-microscopy, DNA barcoding and HPTLC for quality assessment of berberine containing botanicals traded as Maramanjal/Daruharidra

Abstract

Background

Daruharidra/Maramanjal is one of the most popular shrub used in Ayurveda, Siddha and other Indian medicinal systems. More than one botanical source is traded under this name, predominantly Berberis aristata and Coscinium fenestratum with an annual trade of 1000–2000 metric tonnes. The herbal drug trade is often reported with misidentification, adulteration and/or substitution issues due to morphological resemblance and confusion in vernacular names. This work aimed to integrate macro-microscopic, DNA marker strategies and phytochemical assay to differentiate Berberis aristata from its traded sources.

Material and methods

Thirteen marketed samples and one authentic field sample from natural habitat were collected from various regions of the Indian market under the trade name Maramanjal/Daruharidra. The traditional identification methods included macro-microscopic and phytochemical screening by High-Performance Thin Layer Chromatography (HPTLC). Additionally, DNA barcode-based molecular identification and phylogenetic analysis were done using the ITS2 (Internal Transcribed Spacer 2) marker.

Results

The macroscopic observations revealed 80 % ad-mixing of various allied botanicals in addition to accepted north Indian and south Indian sources such as B. aristata and C. fenestratum respectively. DNA barcoding enabled the identification of genuine and adulterated raw drugs from the collected samples. The HPTLC quantification revealed the presence of berberine in all 14 samples varying from 1.12 % to 26.33 %.

Conclusions

The macro-micro, HPTLC, and DNA barcoding helped in the identification of adulteration and substitution practices in this highly traded botanical drug. DNA barcoding can prove an effective tool for discovering the adulteration and substitution of Maramanjal/Daruharidra and this is its first report on the application of morphology, microscopy, phytochemical analysis, and DNA markers in differentiating these traded species.

1. Introduction

Medicinal plants and herbal supplements play an important role in daily life and have long been utilized in the healthcare system. The outbreak of the COVID-19 pandemic has brought enormous attention to the traditional medicine system and the market for herbal drugs is expanding steadily worldwide [1].However, the main issue is the inclusion of adulterants in original herbal drugs due to mismatch in demand and supply. The substituted drugs and adulterations pose substantial risks that must be addressed as they may not provide the promised benefits and sometimes may risk life of the consumer. The primary cause of such confusion can be attributed to the usage of the same vernacular names for plants which are totally unrelated by their phytochemical composition. Furthermore, it can occasionally be challenging to identify plants using conventional methods, particularly when the material is derived from processed plant parts or traded in powdered form [2].The World Health Organization monitors quality control for herbal medication at the global level, as the problem of availability of herbal drugs with assured quality is omnipresent [3].Each country has developed its own pharmacopeial standards for the quality of herbal drugs, but inadequate stringent tests in the monographs on quality standards created by regional regulatory organizations may be one of the reasons for herbal drug non-acceptance [4].In India's traditional medicinal system, various monographs or quality standards have been developed by Pharmacopeia Commission for Indian Medicine and Homeopathy (PCIM&H) [5].

The genus Berberis is represented by 600 species belonging to 12 genera, of which 77 have been reported from India [6]. A yellow spinous shrub belonging to the family Berberidaceae, Berberis aristata is distributed throughout the sub-Himalayan regions and Nepal, and is commonly known as Barberry, Chitra, Daruharidra, and Daru haldi [7]. Daruharidra is a versatile herbal medicine used in Indian traditional medical systems such as Ayurveda, Siddha, and Unani.

B. aristata grows up to a height of 3 m and possesses a woody hard terete stem with brownish-yellow bark outside and yellow wood inside; spines are 3–5 and can be easily removed. Berberine is an isoquinoline alkaloid found in the root, rhizome and stem bark of the members of one of the most primate families [6,8,9].Studies revealed that Berberis spp, such as B. asiatica Roxb ex DC., B. chitria Ham, ex Ker Gawl., B. lycium Royle, B. tinctoria Leschen, B. umbellata Wall. Ex G. Don, and their parts are traded under the same vernacular name Daruharidra in several areas of our country. Similarly, Coscinium fenestratum is traded in markets of South India due to its similar vernacular name ie Maramanjal. There is difficulty in differentiating the wood of these allied species, owing to its yellow color and hard texture, from the official drug B. aristata [10].B. tinctoria and C. fenestratum occur in southern India and the other Berberis spp. occurs in the northern region of India. Berberis spp. is spiny, deciduous, and evergreen while, C. fenestratum, a Menispermaceae member, is a large woody climber and is aberrantly used as a substitute for Berberis spp. Dried samples of the wood are morphologically similar to Berberis, but can be discriminated by the presence of a sclerenchymatous crenate ring below the cortex, lack of annual rings, and wood with large vessels [11,12]. Traditionally, this plant stem has been used for curing acute diarrhoea, amoebiasis, cholera, metabolic disorders and eye diseases [18,19]. Berberis species are widely used for the treatment of inflammation, eye sores, asthma, swelling, drying ulcers, bleeding piles, toothaches, enlarged liver and spleen, urinary disorders, such as painful micturition, skin pigmentation and jaundice [20,21]. Studies on berberine have reported numerous pharmacological properties, such as antioxidative, immunomodulatory, cardioprotective, renoprotective, and hepatoprotective effects [22]. In the recent decade, DNA barcoding has been introduced as an effective tool for the identification of species, in which an unknown nucleotide sequence is matched with the sequence of a known species according to the short fragment information [[23], [24],25]. DNA barcoding of herbal drugs has raised the awareness of substitution as well as adulterants, highlighting the issues in the global market [26]. In the case of land plants, there is no single, universal barcode candidate, while several propositions have been made for using the best barcode candidate for planat species identification [25,27]. The suggested core barcode candidates are matK and rbcL of the plastid genome and the supplementary candidates are the intergenic spacer trnH-psbA (plastid) and nuclear ITS2. In the last decade, advances in sequencing technology like next-generation sequencing (NGS) and whole plastid genome have been suggested as barcodes [28,29]. Few reports revealed that the phylogeny of genus Berberis based on ndhF and ITS loci failed to resolve the species boundaries [30,31].

In the light of challenges of this complex group with the same vernacular names of Berberis species (Maramanjal/Daruharida), we proposed a combined strategy to investigate morphological, macro-microscopic examination, DNA barcoding with HPTLC to distinguish Berberine containing botanicals in the herbal trade.

2. Material and methods

2.1. Collection and identification

The authentic sample was collected from Ranikhet, Uttarakhand, India. Thirteen dried samples were collected from herbal raw drug markets of the country which were sold in the market in the name of Daruharidra/Maramanjal in various forms like stem pieces with and without outermost bark, chipped pieces, and also coarsely powdered. The collected samples were authenticated at the Department of Pharmacognosy, Siddha Central Research Institute (CCRS, Ministry of Ayush), Chennai, Tamil Nadu, India. The voucher specimens of the samples collected were deposited in the raw drug repository of the department to serve as Botanical Reference Material (BRM). Morphological key characters of the authenticated sample were documented using Nikon D-5600 Digital camera and the organoleptic characters were also recorded [32]. Authenticated samples were used for developing the reference standards based on microscopy, DNA barcoding, and HPTLC.

2.2. Macro-microscopy

Pharmacopoeial parameters like organoleptic, macroscopic, and microscopic (transverse section) analysis were done as per Siddha Pharmacopoeia of India [12]. The dried wooden samples were preserved in formalin acetic acid for 48 h before sectioning. The preserved specimens were cut into thin transverse sections using a sharp blade. The sections were stained with 1 % saffranine solution and mounted in 10 % glycerine. The stained sections were photographed using an Axiolab5 trinocular microscope (USA) attached with Zeiss Axiocam208 color digital camera under bright field light. Magnifications were indicated by scale bar. A small amount of the powdered sample was mounted on a microscopic slide with a drop of 50 % glycerol after clearing with a saturated solution of chloral hydrate. Characters were observed using a Nikon ECLIPSE E200 trinocular microscope attached to Zeiss ERc5s digital camera under field light. Photomicrographs of diagnostic characters were captured and documented [32].

2.3. DNA barcoding

2.3.1. DNA extraction

The genomic DNA was isolated from market samples as well as the reference standard using the modified Cetyl trimethyl ammonium bromide (CTAB) method with minor modifications [33]. About 0.5 g of dried samples were crushed into powder using a mortar and pestle and homogenized with liquid nitrogen followed by the addition of pre-warmed 2 ml CTAB (2 %) buffer and 30 μl β-mercaptoethanol. The suspension was transferred to a 2 mL centrifuge tube and incubated in a water bath at 65 °C for 20–30 min with intermittent mixing every 15 min. After incubation, the tubes were centrifuged for 12 min at 12000 rpm using ultracentrifuge (Eppendorf, Germany). An equal volume of chloroform: isoamyl alcohol (24:1) was added to the supernatant, mixed vigorously, and centrifuged for 12 min at 12000 rpm at room temperature. The clear aqueous phase was then collected and transferred into empty centrifuge tubes, and an equal volume of ice-cold isopropanol was added and incubated overnight at −20 °C. After incubation it was centrifuged at 12000 rpm for 12min, the supernatant was discarded, 200 ml of 70 % ethanol was used to wash the pellet and centrifuged at 12000 rpm for 3 min. The DNA pellet was air-dried and re-suspended in 30 μl of Tris-EDTA buffer. The isolated genomic DNA was checked by 1 % agarose gel for electrophoresis run at 5V/cm. In addition, the quantity and purity of the DNA were evaluated using NanoDrop (Nanodrop One, Thermo Scientific, USA).

2.3.2. PCR amplification with ITS2 primer

Total genomic DNA was used as a template, and the PCR reactions were performed for ITS2 (nrDNA) in a thermal cycler (Thermo Fisher, USA). The primer sets and reaction conditions used are listed as supplementary data Table 1A. The PCR assay was carried out using 1 μl (50 ng) genomic DNA as a template, 8 μl EmeraldAmpMAXPCR master mix (Takara, Japan), 5 p.m./μl forward and reverse primers, and the volume was made up to 25 μl with nuclease-free water. The PCR included initial denaturation at 95 °C for 5 min, initial denaturation of 35 cycles at 95 °C for 2 min, the annealing temperature of 55 °C for 30 sec, and extension at 72 °C for 90 sec with a final extension at 72 °C for 10 min [34]. The quality check of amplified regions was carried out with gel electrophoresis on a 1 % agarose gel stained with 1 g/ml ethidium bromide using 1X TAE buffer. The isolated amplicons were eluted using a Qiagen PCR Clean-up Kit, Germany to eliminate potential contamination. The amplified PCR products were purified and sequenced on a 3730XL automated DNA Sequencer by Bioserve Biotechnologies Pvt. Ltd., Telangana, India. Sequencing was done with both primers to retrieve the entire length of the desired region.

Table 1.

Macroscopic observation of samples studied.

SN	IMR Code	Place of collection	Appearance	Color	Odor	Remarks	Probable source
1	IMR2021C1	Gandhinagar, Gujarat	Longitudinally cut wood pieces, 1.5 to 2 in long, 0.25 to 0.5 in thick	Light yellow	Ethyl acetate/acetone-like	Exhausted wood pieces	Unidentified
2	IMR2021C2	Ottapalam, Kerala	Entire stem pieces, up to 2 in long, 0.5 to 1 in dia	Bark greyish, wood bright yellow	Pleasant	Entire stem pieces	Matching to Coscinium fenestratum
3	IMR2021C3	Cherppulassery, Kerala	Bark pieces with thin layers of wood attached to some pieces, slightly curved inwards, up to 3 in long, 0.5 to 1 in wide	Outer surface grey, inner surface bright yellow	Characteristic	Good quality bark	Matching to Berberis tinctoria
4	IMR2021C4	Ooty, Tamil Nadu	Bark pieces with thin layers of wood attached to some pieces, slightly curved inwards, 2 to 3 in long, 0.25 to 0.5 in wide	Outer surface grey, inner surface bright yellow	Characteristic	Entire bark	Matching to Berberis tinctoria
5	IMR2021C5	Ottapalam, Kerala	Longitudinally cut wood pieces, 1.5 to 2 in long, 0.25 to 0.5 in thick	Yellowish brown	Ethyl acetate/acetone-like	Exhausted wood pieces	Unidentified
6	IMR2021C6	Ranikhet, Uttarakhand	Longitudinally split stem pieces, 3 to 4 in long, up to 0.5 in thick	Pale yellow	Nil	Field collected authentic sample	Matching to Berberis aristata
7	IMR2021C7	Kasaragod, Kerala	Chipped stem pieces, 0.5 to 1 in long, 0.25 to 1 in thick	Bark light brown wood brownish yellow	Pleasant, maybe due to adsorption of odor from other botanicals	Good quality stem pieces	Matching to Coscinium fenestratum
8	IMR2021C8	Hyderabad, Telangana	Wood pieces, some with attached bark, up to 1 in thick, 2 in long	Light yellow	Pleasant	Entire wood pieces	Matching to Berberis lycium
9	IMR2021C9	Mumbai, Maharashtra	Coarse powder of wood, longitudinally cut fragments, 1 mm to 1 cm long	Light yellow	Ethyl acetate/acetone-like	Exhausted wood powder	Unidentified
10	IMR2021C10	Chennai, Tamil Nadu (1)	Wood pieces, few pieces with attached bark, up to 3 in long, 2 in wide flat	Bark yellowish-grey, wood brownish-yellow	Mild and pleasant	Entire wood pieces	Matching to Berberis lycium
11	IMR2021C11	Chennai, Tamil Nadu (2)	Longitudinally cut wood pieces, 1.5 to 2 in long, 0.25 to 0.5 in thick	Greenish yellow	Ethyl acetate/acetone-like	Exhausted wood pieces	Berberis sp
12	IMR2021C12	New Delhi, Delhi	Bark pieces with thin layers of wood attached to all pieces, flat, up to 3 in long, 1 to 1.5 in wide, some root pieces also found	Bark grey, wood bright yellow	Characteristic	Good quality bark and wood pieces	Matching to Berberis asiatica
13	IMR2021C13	Palayamkottai, Tamil Nadu	Stem pieces with major area of bark peeled, up to 4 in long, 0.75 in thick	Greyish brown externally, transversely cut surface light yellow	Pleasant	Stem pieces with barks attached at some portions	Unidentified
14	IMR2021C14	Nagercoil, Tamil Nadu	Longitudinally split wood pieces, 0.5 to 1.5 in long, some pieces of barks also, 2 mm to 1 cm thick	Bark grey, wood light yellow, some wood pieces dark yellow	Mild and pleasant	Wood pieces that may be exhausted	Unidentified

2.3.3. DNA sequence alignment and phylogenetic reconstruction

Sequences were initially edited and assembled using Codon Code Aligner version 8.1 (Codon Code Corporation, USA) as this software automatically removes the low-quality sequence at the start and end of the sequence. The Phred score 20 or above was set for the quality check of the sequences. Both forward and reverse primers are trimmed from the assembled sequences and the edited sequences were then aligned using MUSCLE algorithm in the MEGA 11 package [35]. BLAST analyses were performed for all assembled sequences obtained from GenBank to check the potential contamination with unknown species and the threshold value of 99 % identity was set for the top match [36]. The Basic Local Alignment Search Tool (BLAST) from the National Centre for Biotechnology Information website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was then used to compare these sequences with in-house sequences and GenBank database sequences [37].

The multiple sequence alignment was analysed in ClustalW within MEGA 11 software to calculate the genetic distance between accessions, eliminating all the positions with missing data using Kimura's 2-parameter model. The phylogenetic tree construction was achieved through the Maximum Likelihood (ML) method with 1000 replicate bootstrap. To estimate the resolution of the ITS2 barcode, the percentage of monophyletic groups generated was calculated using a bootstrap higher than 80 % as a parameter to define the nodes [38].

2.4. HPTLC analysis

2.4.1. Chemicals and solvents

The chemicals viz., n-hexane, chloroform, ethanol used for isolation were of laboratory grade and procured from Reachem Chemicals, Chennai, India; and that of quantification were of Analar grade solvents viz., toluene, n-butanol, ethanol, ethyl acetate, acetic acid and formic acid and purchased from Merck Millipore, Munhal, Mumbai, India.

2.4.2. Isolation of berberine

Ethanolic extraction was carried out using 100 g of the samples by cold percolation method yielding 5 g. It was column chromatographed (60 cm long; 2.5 cm diameter glass column) over silica gel (Acme's 60–120 mesh). It was first eluted with n-hexane and all the fractions were combined and kept separately. Further, it was eluted with chloroform: ethanol in varying compositions. The fractions eluted with chloroform: ethanol (80:20 to 50:50, v/v) afforded a yellow-colored compound (56 mg). It was recrystallized from methanol to get 50 mg of pure compound. It gave a single spot at R_f 0.57 with the mobile phase n-butanol: ethyl acetate: acetic acid: water (3:5:1:1, v/v/v/v). It's UV spectrum showed an absorption max at λ265, 348 [39,40] and its melting point was observed as 145 °C which matched the literature value [41].

2.4.3. Preparation of standard solution

Berberine (1 mg) isolated from authentic Berberis aristata was dissolved in 10 ml of ethanol to make a solution of 1 mg/10 ml of standard. For weighing, a 4-decimal analytical digital balance (Sartorius-Quintex 224-10IN) was used.

2.4.4. Preparation of sample solutions

The samples were ground in mortar and pestle and homogenized into coarse powders. Powdered samples (1 g) were extracted with 100 ml ethanol using Soxhlet apparatus, filtered through Whatman no. 42 filter paper, concentrated, and made up to 10 ml in standard flasks.

2.4.5. Preparation of working solutions of sample

For the test samples IMR2021C5, C6, C9, C11, C13, and C14 the above stock solution of 1g/10 ml was used. For samples, IMR2021C1, C2, C7, C8, C10, and C12, the 1g/10 ml stock solution was double diluted viz. 5 ml diluted to 10 ml resulting in 1g/20 ml. For samples IMR2021C3 and C4, 4 ml of the stock solutions were diluted to 10 ml resulting in 1g/25 ml. The aforesaid concentration were fixed considering the content of berberine after preliminary TLC trials.

2.4.6. HPTLC instrumentation

For the application of standard and sample solutions, silica-coated aluminum TLC plate 60F₂₅₄ (Merck) of 0.2 mm thickness and Camag Linomat 5 applicator were used. For developing the TLC plate, a twin trough chamber (CAMAG 20 × 10 cm) was utilized. The developed plate was photo-documented using CAMAG TLC Visualizer under UV light (254 nm, 366 nm) and after spray with Dragendorff's reagent. For generating fingerprint profiling and quantification, a TLC densitometric scanner equipped with winCATS software (version 1.4.2) was used.

2.4.7. Berberine estimation procedure

Before the application of samples, the TLC plate was prewashed using methanol solvent and activated at 105 °C in a hot air oven for 5 min. The standard solution was applied in six tracks with volumes 2, 4, 6, 8, 10 and 12 μl equivalent to 2000–12000 ng. Based on the trial runs of the quantification, different volumes of samples were applied to bring them within the linearity range. For samples IMR2021C1, C2, C5, C7 to C13, a volume of 3 μl of each sample was applied. For samples IMR2021C3, C4, C6 and C14, the applied volume was 5 μl on the TLC plate (20 × 10 cm) as 6.0 mm bands at a distance of 6 mm between the tracks and application position of 10.0 mm on the Y axis and 15 mm on the X axis. A linear ascending chromatographic development was carried out up to a distance of 80 mm at a temperature of 27 °C with toluene: ethyl acetate: methanol: formic acid (6:5:2:1, v/v) as the mobile phase. After chromatographic development, the TLC plate was dried, and the photos were taken at 254 nm and 366 nm [42] and scanned in absorption mode at 265 nm using a deuterium (D₂) lamp. The slit dimension of 5 × 0.45 mm was selected and scanned at a rate of 100 nm/s. After that, the plate was derivatized with Dragendorff's reagent. The alkaloids appeared as orange-colored spots which were immediately photo-documented under the white light.

3. Results

3.1. Macroscopic characters

A morphological comparison of the market samples and authentic Berberis aristata indicated that at least 80 % of market samples are admixed with similar looking botanicals. From the observations of the collected samples, the raw materials were categorized into three groups viz. Berberis, Coscinium and unidentified species based on their external morphology (Fig. 1); the macroscopic details are summarized in Table 1.

Fig. 1.

Morphology of the samples studied.

The morphological evaluation provided mixed results and we were unable to conclusively determine whether the samples are authentic or not. Further, we proceeded to evaluate the applicability of microscopy, DNA barcoding, and HPTLC to determine the authenticity and quality of the market samples.

3.2. Identification by microscopic characters

The microscopic observation led to the identification raw drugs belonging to two genera namely Berberis (7 samples, 4 species) and Coscinium (2 samples, 1 species) together with some unidentified species (5 samples) as described below (Fig. 2, Table 2).

Fig. 2.

Microscopy of stems of the samples studied.

Table 2.

Microscopic observations of the samples studied.

SN	IMR Code	Observation	Identified as
1	IMR2021C1	The anatomy of the sample does not match with Berberis sp and C. fenestratum	Unidentified
2	IMR2021C2	Anatomy matching with C. fenestratum	Coscinium fenestratum
3	IMR2021C3	The anatomy of the sample does not match with B. aristata and C. fenestratum	Berberis tinctoria
4	IMR2021C4	The anatomy of the sample does not match with Berberis sp and C. fenestratum	Berberis tinctoria
5	IMR2021C5	The anatomy of the sample does not match with B. aristata, B. asiatica and C. fenestratum	Unidentified
6	IMR2021C6	The anatomy of the sample matching with B. aristata	Berberis aristata
7	IMR2021C7	Anatomy matching with C. fenestratum	Coscinium fenestratum
8	IMR2021C8	The anatomy of the sample does not match B. aristata and B. asiatica; maybe B. lyceum	Berberis sp
9	IMR2021C9	The anatomy of the sample does not match with Berberis sp and C. fenestratum	Unidentified
10	IMR2021C10	The anatomy of the sample does not match with B. aristata and B. asiatica	Berberis sp
11	IMR2021C11	The anatomy of the sample does not match with B. aristata and B. asiatica	Stem and root of Berberis lycium
12	IMR2021C12	The anatomy of the sample does not match with B. aristata and B. lyceum; maybe B. asiatica	Berberis sp
13	IMR2021C13	The anatomy of the sample does not match with Berberis sp and Coscinium	Unidentified
14	IMR2021C14	The anatomy of the sample does not match with Berberis and C. fenestratum	Unidentified

3.2.1. Berberis

The transverse section of the stem showed an outer well-developed cork made up of lignified rectangular cells containing suberin depositions; the cortex composed of tangentially elongated parenchymatous cells with randomly scattered groups of stone cells; narrow phloem made up of sclerenchyma and fibers and is surrounded by a discontinuous ring of pericyclic fibers; distinct cambium separates the phloem from xylem; medullary rays are seen traversing through the xylem region; xylem occupies the remaining portion of the section. The powder microscopy showed the presence of thick walled parenchyma, pitted tracheids, spiral and reticulate vessels, sclereids, stone cells and yellow contents (Table 2A).

The macro-microscopical observation of the samples led to the identification of 4 different species of Berberis namely B. aristata, B. asiatica, B. lycium, and B. tinctoria. The major anatomical differences observed are as follows.

• aBerberis aristata: Xylem vessels numerous, small to medium-sized, in single or in groups arranged radially; medullary ray in continuation with xylem and containing calcium oxalate crystals; dark brownish content found occasionally in ray cell (Fig. 2.6).
• bBerberis asiatica: The vessels are arranged in a diffused semi-ring porous pattern; the vessels are oval and medium-sized; medullary rays are uni to multiseriate; the xylem consists of vessels, tracheids, fibers, and parenchyma (Fig. 2.12).
• c
  (i) Berberis lycium stem: Xylem vessels are few and arranged diagonally and diffused pattern; vessels are oval to angular smaller in size when compared to B. aristata and B. asiatica; vasicentric tracheids in between vessels, libriform fibers filled with starch grains, multiseriate rays 5 to 10 cells wide (Figs. 2.8, 2.10 and 2.11A).

  • (ii)
    Berberis lycium root: Xylem vessels are arranged in a semi-ring pattern occurring mostly in diagonal groups, the number of vessels per group of 12–15. Vasicentric tracheids are abundant and found intermixed with vessels: libriform fibers with starch grains; mostly multiseriate rays of 6 to 8 cells wide; few uni to biseriate (Fig. 2.11B).
• dBerberis tinctoria: Xylem vessels few, very smaller in diameter when compared to all other species; vessels single or rarely in group of 2; phloem region broad; bi to multiseriate medullary rays traversing through the vascular region; large parenchymatous pith (Figs. 2.3 and 2.4).

3.2.2. Coscinium fenestratum

TS of the stem showed an outer cork made up of 20–30 rows of suberized thick-walled rectangular cells filled with some yellowish content; the cortex is very narrow traversed with few stone cells and patches of fibers; the pericycle is distinct and continuous and forms an arc surrounding the vascular bundles; phloem consists of sieve tubes, companion cells and parenchyma; medullary rays are wedge-shaped made up of thick-walled, multicellular pitted cells; xylem occupies the major portion of the section (Figs. 2.2 and 2.7). The powder microscopy showed the presence of cork fragment, parenchyma cell, pitted and bordered pitted vessels, tracheids, sclereids and stone cells (Table 2A).

3.2.3. Unidentified

The TS of the remaining samples differed in anatomy from both Berberis and Coscinium characters (Figs. 2.1, 2.5, 2.9, 2.13, 2.14).

3.3. Identification by DNA barcoding

Reference DNA barcodes were developed from authentic Berberis using the ITS2 barcode marker. The success rate of ITS2 primer used for PCR amplification was 43 % with an average sequence length of 340 bp (Fig. 3). Out of 14 samples, Kasaragod sample was identified as Coscinium fenestratum; Ranikhet sample as Berberis aristata; Hyderabad as Berberis lyceum; and New Delhi as Berberis asiatica by BLAST analysis. All sequence amplicons of the Berberis and its adulterant were deposited in the GenBank (Table 3). The remaining samples didn't have satisfactory PCR amplification which might be due to the age of the dried samples because the majority of market samples were dried and exhausted and yielded a minimal quantity and low quality of genomic DNA; there may be breakage in the invariant primer potential region in their extracted DNA leading to reduced amplification efficiency, fragmentation, or nonspecific amplifications. To evaluate whether species were recovered monophyletic under Berberis species, a phylogenetic tree was constructed by Maximum Likelihood (ML) with Kimura 2- parameter + gamma-distributed rate best-fit model based on ITS2 DNA barcode sequence from Berberis aristata and the market samples' DNA which gave amplification. The number of nodes indicated bootstrap values with 1000 replicates.

Fig. 3.

Gel image of PCR amplified products.

Table 3.

The list of species identified by BLAST analysis.

SN	Voucher ID	Plant name	Location	Collection date	Genebank ID
1	IMR2021C6	Berberis aristata	Ranikhet	30-04-2022	OR765726
2	IMR2021C7	Coscinium fenestratum	Kasargod	28-01-2022	OR764596
3	IMR2021C8	Berberis lycium	Hyderabad	16-02-2022	OR754367
4	IMR2021C12	Berberis asiatica	Delhi	29-11-2021	OR742177

There was a total of 353 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [40] in C. fenestratum, B. aristata, B. asiatica and B. lycium (Fig. 4). The tree phylogram included authentic B. aristata samples and commercial samples. The nucleotide sequence of C. fenestratum were utilized as an outgroup which enhances the understanding of genetic relationships of Berberis commercial samples. The results revealed that all the Berberis samples were clustered within the same clade such as B. aristata, B. lycium and B. asiatica and C. fenestratum in another clade. To ensure efficacy, quality, and safety, this study advocates the integration of DNA-based methods, chemical analysis, and microscopic and macroscopic approaches for the authentication of herbal materials used in the herbal industry.

Fig. 4.

Phylogeny of samples identified by BLAST.

3.4. HPTLC

Determination of berberine was performed for all the collected samples. The TLC solvent system was fixed based on trials conducted to fix the appropriate concentration with linearity. The identity of the marker was confirmed by the value of R_f and absorption maxima of 265 nm. The R_f value in the range of 0.55 %–0.57 % confirmed the presence of berberine in all the examined 14 samples (Fig. 4). Linear calibration graphs of peak area vs. concentrations were created to calculate the coefficient of correlation (r), standard deviation (SD), and linear regression analysis of the compounds. LOD and LOQ was 0.0029 μg and 0.0088 μg respectively (Fig. 5). The HPTLC quantification revealed the presence of berberine in all 14 samples varying from 1.12 % to 26.33 %. Berberine concentration was the highest in the sample collected from Ooty (IMR2021C4) while the lowest concentration was present in the sample collected from Ranikhet (IMR2021C6) (Fig. 6 and Fig. 7).

Fig. 5.

HPTLC images of samples studied.

Fig. 6.

Estimation and quantification of Berberine.

Fig. 7.

Linearity range and Calibration graph.

4. Discussion

The confusion of herbal drugs with the same vernacular name may impact consumer safety and lead to undesirable effects. Due to lack of standardization and quality control profiles the search for definitive methods for the authentication of herbal drugs is widely explored by quality control experts [43]. Challenges faced in the herbal drug market due to the vernacular name confusion has been reported more frequently. In Thailand, a traditional herbal formula consisting of Mallotus repandus (Willd.) Mull. Arg. commonly known as “Kho-Khlan” is used for pain relief. Two other plants namely Anamirta cocculus (L.) Wight and Arn and Croton caudatus Gleiseler also share the common name. The usage of A. cocculus or C. caudatus have effects via toxicity or unsuccessful treatment [44]. Similarly two popular vegetables named Melientha suavis Pierre and Sauropus androgynus (L.) Merr. also shares a common vernacular name, “Phak Wan”, with a poisonous plant species Urobotrya siamensis Hiepko. The unintentional consumption of U. siamensis resulted in comas and deaths in 2005 [45]. A few reports state that species identification of Berberis harvested during winter months becomes difficult due to similar phenological characteristics among related species [46,47].

In the last few decades, several reports have been published on the successful application of taxonomic identification, DNA barcoding, and HPTLC analysis for the authentication of herbal materials. However, the presence of similar vernacular names, similar morphology, and overlapping metabolites limits the applications of these methods. The present study developed a strategy to identify adulterants from the market samples having similar morphology and similar vernacular names using macro-microscopic characters and DNA barcoding coupled with the HPTLC fingerprinting. Daruharidra/Maramanjal, botanically equated to Berberis aristata L. as the official source, is a potent medicinal plant finding use in almost all traditional medicinal systems, and it is sold all over the country as a raw material for preparation of many medicines in Indian Systems of Medicine. The most striking feature of this drug is the presence of isoquinoline alkaloid, berberine, which is found in the root and stem bark imparting it a yellow color. As the alkaloid is of numerous therapeutic efficiency there is a high demand for this drug. In south India Coscinium fenestratum locally known as Maramanjal is sold in the market instead of B. aristata which also contains abundant berberine. Unaware of the botanical identity, the common man buys all the botanicals traded the market. We collected raw drug samples from various herbal vendors throughout the country in the same vernacular names. The macroscopic observation led to the identification of an admixture of samples belonging to various genera namely Berberis, Coscinium, and some unidentified species. The genus Berberis and Coscinium can be differentiated based on the transverse cut surface of stem showing a wheel-like appearance in the latter [48]. From the market survey it was found that some spurious samples that are yellowish in color are available in the market and are difficult for the untrained eye to identify owing resembling organoleptic characteristics.

The microscopic observations led to the identification of various species of Berberis from the arrangement of xylem vessels and their density. From the total of 14 collected samples, 7 samples belonged to Berberis species and 2 samples to Coscinium fenestratum. Leading from the difference observed anatomically 4 different Berberis species could be demarcated namely Berberis aristata, B. asiatica, B. lycium, and B. tinctoria. However, these 4 species were hardly distinguished by their morphology and histology due to close taxonomical relationship. The remaining 5 samples showed marked differences in their wood anatomy and could be ruled out from both Berberis and Coscinium.

Subsequently, we investigated the species resolution ability of all market samples using the ITS2 barcode candidate. There are some mixed results about PCR success and sequencing using ITS2 primers in Berberis species. Despite the successful design of specific genus-specific markers like matK and rbcL in Berberis, their successful rate was 76 % and 85 % [30]. Only 4 out of 14 samples showed the species resolution, which includes B. aristata, C. fenestratum, B. lycium, and B. asiatica. The lower success rate of PCR using other primers in Berberis may be due to the instability and uniqueness of primers in 3'end. A successful barcode is evaluated based on interspecies divergence rather than intraspecies divergence [49,50]. In most of the earlier reports, multilocus analysis of more than 3 loci didn't gain any species recovery in the Berberis genus and the combination of ITS with plastid trnH-psbA was suggested for gaining better species discrimination as compared to other multilocus barcode analyses [30]. DNA barcoding investigations employing markers such as matK, rbcL, and nuclear regions across diverse floristic taxa have revealed a high level of species- level resolution [51]. These loci were not been worked in other studies while dealing with specific taxonomic groups like the Berberis genus. The lowest level of barcoding success observed in the genus Berberis is not uncommon in plants. Similar kinds of difficulties have earlier been reported in genus Aspalathus, Crocus, Solanum sect, Petota, and Corex, even in the well-studied taxonomic group of Hordeum [30]. In the case of Berberis species earlier studies have discussed that different species of Berberis namely B. asiatica and B. lycium are used as adulterants of B. aristata [52]. C. fenestratrum is used as the source of Maramanjal in south India, though not an official source, and is used in the preparation of various classical formulations for the treatment of various ailments along with the roots and woods of different Berberis species [53]. There are nearly 50 NCBI submissions of B. aristata already present in the database [54] and through the present study we have added 4 new submissions to it.

Furthermore, the qualitative and quantitative examination of berberine from all collected samples were carried out by HPTLC. The qualitative examination confirmed the presence of berberine in all the samples. Quantitative estimation showed the highest concentration in the traded market sample of Berberis, viz Berberis tinctoria of 26.33 %, and the lowest concentration was observed in the authentic sample collected from Ranikhet. The reduced concentration in the authentic sample can be attributed to the age of the sample as we had collected it from a young tree. Mikage and Mouri, 1999, have previously reported that the percentage of berberine will be less in species growing in high altitudes when compared with those growing in low altitudes [55]. The variation in the percentage of the alkaloids in the samples studied can be accredited to the geographical locations also. Studies revealed that the roots of the plants possessed a higher berberine content than the stem and stem bark; the wood weighs approximately 620–960 kg/[13]. The berberine content of roots of B. asiatica (4.30 %) is comparatively higher than B. lycium (4.00 %), B. aristata (3.80 %), as per previous reports [[14], [15], [16]]. Researchers also reported a higher amount of berberine (2.8 %) in B. aristata than in B. asiatica (2.4 %) [[7], [17]].The HPTLC profiling of various Berberis species has been performed in the past but there is inconsistency in the percentage of berberine observed in different species in a study by Srivastava et al., 2004 [17] reporting the higher concentration in B. aristata when compared to B. asiatica while Andola et al., 2010 [56] testified B. asiatica to have more percentage than B. lycium and B. aristata. In our study the highest amount of berberine was recorded in B. asiatica followed by B. lycium and the least amount was observed in B. aristata.

Berberine the alkaloid of immense medicinal potency is isolated from the plant species occurring in various families like Rutaceae, Ranunculaceae, Papavaraceae, Menispermaceae, and Berberidaceae [22]. As this natural alkaloid is the precursor for the synthesis of various bioactive derivatives [57] many raw drugs market samples contained lower berberine concentrations as they reached the market after the extraction (exhausted) of berberine. The adulteration of herbal medicines is a burning topic that needs to be resolved at the earliest as it poses a threat to the validation and efficacy of traditional medicines that rely on herbal raw drugs. Visualization of the spurious actions of adulteration and substitution is quite difficult. From the taxonomical identification concerning the macroscopy, to the tissue detailing by anatomy, followed by the chemical profiling, and finally the DNA barcoding, we could differentiate and identify the actual drug and its adulterants. In the case of dried raw drugs, where the PCR amplification and the subsequent sequencing and identification failed, the microscopic study dealing with the anatomical parameters proved to be an excellent tool in their identification. It was found that various botanicals having berberine, which imparts a yellow color were sold in the market as Daruharidra/Maramanjal. C. fenestratum was identified as the adulterant in the southern part of the country while various species of Berberis namely B. lycium and B. asiatica and some unidentified raw drugs were encountered in the samples collected from northern India. Consequently, we could prove the adulteration of Daruharidra/Maramanjal with various other unidentified taxa. Thus, this comprehensive study proved effective in identifying and authenticating genuine drugs and substantiated itself to be effective in the quality control of Berberis.

5. Conclusion

Due to the high demand, limited availability, and confusion in vernacular names, adulteration and substitution in Indian Berberis species are increasing alarmingly. Despite its widespread application, the quality standards for berberine botanicals have yet to be established. The standardization of herbal drugs requires advanced analytical methods using state-of-the-art instrumentation. The first and most crucial step in herbal drug research is developing quality standards, as a herbal product without defined standards may lack the required efficacy. Macro and microscopic assessment effectively distinguishes all the 14 market samples which includes B. aristata, B. asiatica, B. lycium, B. tinctoria, C. fenestratum, Berberis sp., and some unidentified species. In addition, DNA barcoding of the ITS2 region was effective in differentiating the market samples. Moreover, HPTLC fingerprinting detected distinctive bands among the Berberis species and other adulterant species, inferring all the botanicals are in trade due to the presence of berberine in it and hence the yellow color. The integrated analysis of macro-microscopic examination, DNA barcoding, and HPTLC revealed the identity of all market sample trades as Daruharidra/Maramanjal. This study emphasizes the effectiveness and robustness of integrating these techniques for authenticating and ensuring the herbal materials/plants in the industry as well as in the market. There are individual advantages and disadvantages of each method, but, a comprehensive approach employing all technologies could enhance the accuracy and efficiency in the authentication of raw drugs. This kind of study is essential for ensuring the safety, efficacy, and quality control of herbal medicines.

Author contributions

KNS Conceptualised the project and obtained the grant as Principal Investigator. KNS, KGD and PR are involved in the project execution. KM, PK and AR performed the molecular experiments and contributed to manuscript writing; SS conducted the microscopic characterisation; MS and SR conducted the HPTLC studies; KM, KGD, KNS, and SR done the manuscript writing to the publishable format. AK contributed to the Siddha aspects of the drug.

Declaration of generative AI in scientific writing

Nil.

Sources of Funding

This work was funded by Central Council for Research in Siddha, Chennai, Tamil Nadu, Ministry of Ayush, Govt. of India as an Intra Mural research project (F.No:1–67/2017-CCRS/Tech/IMR382/(1) dated 19 March 2020).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.