Identification and authentication of Agnimantha plant species used in Ayurveda on the basis of anatomical and molecular phylogenetic analysis

Agnimantha plant species have been used in the Ayurvedic system of medicine for many years and is widely used as an ingredient in many ayurvedic formulations. However, the source for Agnimantha remained controversial as it is difficult to authenticate from various reports. Hence, the present study aims to identify and authenticate its original and substitute sources. As per the literature sources Clerodendrum phlomidis L.f. , C. inerme (L.) Gaertn. and Premna serratifolia L. are considered Agnimantha species. The anatomy of the above mentioned species confirmed the presence of patches of up to 20 cells in the sclerenchyma of the root cortex, while in the absence of sclerenchyma of the stem cortex, abundant chambered crystals were also present in the bark of the stem and root in C. phlomidis as compared to C. inerme and P. serratifolia . Phylogenetic analysis using chloroplast ( matK, trnH - psbA ) and nuclear markers ( ITS, rbcl ) also indicates the close relation between C. inerme and P. serratifolia and hence places them both in the same clade, though C. phlomidis is closely related to the other species but placed in the adjacent clade. Hence, the study concludes that anatomical as well as molecular phylogenetic analysis reflect close relation between C. inerme and P. serratifolia . while a distant relation with C. phlomidis.


Introduction
The botanical identity of Agnimantha, which is one of the Dashamula plant species, is still controversial. Physicians (ayurvedic) from different regions of India considered the source plants for Agnimantha are Premna serratifolia L., P. tomentosa Willd, P. mollissima Roth, P. coriacea var. villosa (C.B.Clarke) A.Rajendren & P.Daniel, Clerodendrum phlomidis L.f. and C. inerme (L.) Gaertn. (1). There is no description of types of Agnimantha in ancient texts, i.e., Charaka Samhita and Sushruta Samhita. Some Nighantus have described two types -Brihat Agnimantha and Kshudra Agnimantha (2). Premna serratifolia is considered Brihat Agnimantha and C. phlomidis is considered Kshudra Agnimantha, whereas according to the Ayurvedic Pharmacopoeia of India, C. phlomidis is the original source plant to be considered in Agnimantha (3). Moreover, Ayurvedic Formulary of India (Part I) reported C. phlomidis as the authentic plant source of Agnimantha while P. serratifolia Willd and P. mucronata Roxb. are substitutes.
Agnimantha is one of major component in the ayurvedic system of medicines like Brahmarasayana, Chavanaprasha, Narayanataila and Gorocanadivati. In Agnimantha, roots and leaves are used as crude drugs (4). It is also used as ingredient in around 25 products i.e., Dasamula. In the list of 70 medicinal plant species of high trade sourced from tropical forests, roots of P. serratifolia and C. phlomidis, are also included (5). P. serratifolia is considered as threatened red listed species of IUCN (6).
Agnimantha leaves are used to reduce inflammation and pain, in the treatment of Arshsas for Avagaha Sweda (tub bath sudation, Panchkarma) (7). The leaves of P. serratifolia are used in the treatment of colic, rheumatism, neuralgia, etc. Whereas C. phlomidis is used in dyspepsia, dysentery, colic, debility, and neural disorders, and P. latifolia Roxb. has germicidal activity and is used in the treatment of dropsy, colitis, and boils (8,9). The juice of C. inerme leaves is used for reducing muscular pain, rheumatoid arthritis, and stiffness of the legs (10). Documentation, including studies on authenticity and substitute sources for Agnimantha, is inadequately documented. Consequently, this led to a lack of transparency regarding the use of authentic Agnimantha as mentioned in ancient texts. But to be accepted globally along with safe and effective ayurvedic products, identification of the authentic botanical entity is important for regulatory compliance.
The micromorphological method for authentication of herbal formulations and crude drugs is one of the important steps in r standardization. Premna serratifolia, C. inerme and C. phlomidis are different species, but they are collectively known as Agnimantha. Though the taxonomic identification of plants can easily be done by their morphological characteristics, the same is difficult when they are in dried form as many of the characteristics of plant parts change after drying. In case where plant drugs are in crude drug form, molecular and pharmacognostic studies will be highly effective in determining their botanical identity.
Pharmacognostic studies of the root of Agnimantha (P. serratifolia and C. phlomidis) have already been carried out (11)(12)(13)(14). Pharmacognostic studies on the leaves and stem of different sources used as Agnimantha are not available. In this regard, comparative pharmacognostic evaluation of the leaves of P. serratifolia, C. phlomidis, and C. inerme was taken into consideration to authenticate the substitution of these source plants for Agnimantha.
In molecular phylogeny, a short sequence of DNA is used for accurate identification and authentication of species, complementing the classical taxonomic methods (15). Though DNA barcoding has been effectively used for classifying animal species, the same approach should be used to discriminate among plant species. Mitochondrial genomes of plants reveal minimal nucleotide substitution and maximal chromosomal rearrangements. On the other hand, the nuclear genome of plants shows extensive gene duplication. Previous DNA barcoding studies of plants have suggested some plastid coding and non-coding regions, like trnH-psbA, rbcL, matK, rpoC1, and rpoB, as promising candidates (16)(17)(18)(19)(20)(21). Nonetheless, the gradually evolving coding regions in the genome of plastids may not possess sufficient variation to categorize closely related plant species, which might lower their potential as efficient barcodes. This problem can be solved by analysing selected loci either in combination or individually. The recently evolved nuclear region, i.e., the nuclear ITS spacer from the ribosomal ITS gene has also been suggested as a promising barcode.

Plant material collection and storage
Root, stem, and leaf samples were collected from one plant of each species mentioned below available in the Medicinal Plants Garden, Regional Ayurveda Research Institute, Pune, Maharashtra (India). The herbarium of the plant specimens of Clerodendrum phlomidis (SVUBS 2220), Clerodendrum inerme (SVUBS 14151), and Premna serratifolia (SVUBS 4536) was prepared and deposited in the herbarium of the Institute (RARI, Pune). All the plant samples were thoroughly washed under running tap water and fixed in the plant fixative FAA (Formalin, Acetic Acid, Alcohol, 10%:5%:50% + 35% water) before sectioning of plant tissues.

Anatomical analysis
Dehydration of plant tissues was done in an ascending series of ethanol up to 100% for one hour in each ascending series of alcohol using Johansen's technique (22). Infiltration was done by keeping tissues in wax in an oven at 60°C for 1 h, where the wax melted and the samples sank slowly into it. The xylene-wax mixture was then replaced with a fresh amount of pure wax for 30 minutes in the oven. The samples were embedded into pure paraffin wax in moulds and allowed to set for 1 hour to solidify. Properly embedded samples were sectioned at 12-15 µm thickness with a rotary microtome (Wasewox, India). The sections were de-paraffinized and rehydrated in a descending series of ethanol and then stained with Haematoxylin (Himedia, India) and safranin using standard laboratory procedures (22). Each slide was washed in running tap water for 2 minutes, then slides were dehydrated in an ascending series of ethanol and absolute ethanol with gentle shaking and mounted in Canada Balsam (Himedia, India). Microscopic data and photomicrographs of characteristic features were taken from a light microscope (Olympus BX45, Japan) equipped with a digital camera. DNA extraction, amplification and sequencing Extraction of DNA was done from tender leaves using a modified kit-based technique (Qiagen, USA). The absence of a specific region as a universal plant barcode led to consideration of four genomic loci corresponding to matK, rbcl, trnH-psbA, and ITS for developing DNA barcodes based on literature. The sequence information for most of the above-mentioned loci was not available for Clerodendrum and Premna species, attempts were made to amplify three chloroplast and a nuclear DNA marker (Table  1) in three plants to be considered in Agnimantha (C. phlomidis, C. inerme and P. serratifolia). Amplification of the desired locus was accomplished by taking a final volume of 25μL (Table 2) and the amplicons were visualized on 1.2% agarose gel (23). The presence of sharp single bands was taken for further sequencing (24) analysis. Sequencing of the amplified regions was performed using the Sanger Sequencing method (Eurofns Genomics India, Pvt. Ltd. Company).
The extracted DNA was quantified on a biophotometer by taking absorbance at 260 and 280 nm to calculate A (260/280) (Eppendorf Biophotometer Plus, Hamburg, Germany). The concentration of DNA was 25 ng/ μL and O. D was 1.8.

PCR amplification
PCR amplification was conducted using (50-100) ng of genomic DNA as a template in a 25 μL of PCR mixture (Table 1) containing 12.5 μL of PCR master mix (2X), 10 µM forward and reverse primers (2µL each), 3.5 μL nucleasefree water. The PCR conditions for different primer pairs were given below (rbcl and matK (Table 3), ITS (Table 4) and trnH-psbA ( Table 5). The amplified products were detected by 1.5% agarose gel electrophoresis in TAE buffer and visualized by using ethidium bromide on Gel documentation system (BIORAD, USA).

Analysis of DNA sequences
Chromatograms of each and every single sequence were verified, and poor-quality DNA sequence ends was trimmed before making contigs. 60% of the original read length were maintained after trimming the raw sequences, subject to the minimum average quality score of Q20. All single nucleotide variations were assessed and validated by aligning the chromatograms from both forward and reverse sequencing results. Sequences with 80% and above overlap were used for creating consensus sequences for all the amplicons. The sequence was checked using Chromas for specific base pairs and editing and consensus sequence formation Bioedit was used. Suitable sequences from all the individuals were assembled and aligned using CLUSTALW 1.83 (for multiple alignment). Conserved, parsimony, and variable informative sites were determined using MEGA 11. A phylogenetic tree was drawn using the maximum likelihood method through MEGA 11 software. Outgroups   Tectona grandis (MN814870.1) used in phylogenetic tree were obtained from NCBI.

Result
In the present study, molecular and pharmacognostic analysis of three plant species being contemplated as sources of Agnimantha have been carried out. Premna serratifolia, Clerodendrum phlomidis and Clerodendrum inerme belonging to Lamiaceae, are considered source plants for Agnimantha.
On analysis, the transverse section of the leaves showed parenchyma well differentiated into upper palisade and lower spongy tissues, the presence of a cuticle on the epidermis, and anomocytic stomata on the lower surface. The presence of prismatic crystals of Calcium oxalate was observed in two species except P. serratifolia. On analysis of T.S. through the midrib, it showed radially arranged protoxylem towards the upper epidermis and metaxylem towards the lower side of the vascular bundle. A specific microscopic description of the species is given below, along with the sequence analysis of the molecular markers amplified.

Microscopic description Premna serratifolia L.
Stem: A T.S. obtained from the stem showed a nearly circular outline. The epidermis of stems consists of a single layer cell. The secondary cortex is narrow and composed of patches of sclerenchyma alternating with compressed parenchymatous cells (Fig. 1C & 2F), particularly above the phloem. The thin layer of cork cells is composed of elongated lignified cells. The secondary phloem is composed of tangential zones containing sieve elements and companion cells, which alternate with axial parenchyma cells and are permeated by a network of phloem rays. Secondary phloem rays are both uniseriate and biseriate. Both uniseriate and multiseriate rays are composed mostly of square and upright cells throughout the body. Tangential multiples of 2-3 stone cells are also present in the secondary phloem after some intervals ( Fig.  1C & 2F). Secondary xylem comprises rounded to ovalshaped vessels that are mostly solitary with a few radial multiples of 2-3 ( Fig. 1A & C). The vessel diameter is 30 (20-40) um and the vessel length is 350 (250-500) µm. Perforation plates in vessels are simply rounded to oval with distinctly bordered alternate pits on their lateral walls ( Fig. 1F & G). The axial parenchyma is scanty paratracheal. Fibers are thin-walled, septate with simple pits on radial walls. Rays are uniseriate to biseriate, composed of square and upright cells only throughout the body (Fig. 1E). Vessel ray pits are simple to round with a reduced border. Any kind of crystal or mineral deposition was not found in both xylem and phloem. A narrow pith made of parenchymatous cells is also present, followed by secondary xylem.

Root:
The transverse section of the root is circular in outline, consisting of wavy cork cells followed by the cortex and vascular zone (Fig. 1B). The Cork layer is relatively thick in 4-6 layers of cells. The cortex is narrow and made up of thin-walled parenchyma. The xylem is well -developed and broader, measuring about three-fourths of the diameter of the cross-section. Xylem vessels with rounded to oval outlines are mostly solitary with few radial multiples of 2 or 3. The vessel diameter is 45 (25-70) um (Fig. 1D). Rays are conspicuous, both uniseriate and biseriate. Pith is absent in the mature stage of the root.
Leaf: A transverse section of the petiole and midrib is composed of a single layered epidermis and cuboidal cells with cuticle thickening. The upper epidermis is followed by collenchyma cells (3-4 layers), and the cortical tissue is composed of numerous rows of parenchyma cells. Vascular bundles containing xylem and phloem were present in the centre of the midrib. The phloem is composed of parenchyma, sieve tubes, phloem rays, and phloem fibres. The xylem is composed of xylem vessels, fibers, wood parenchyma and xylem rays. Wood parenchyma containing elongated cells with pitted walls and spiral thickening was present on xylem vessels ( Fig. 2A  & C). At the centre, the ground tissue is made of parenchyma. The metaxylem occurs radially towards the periphery (abaxial surface), while the protoxylem occurs radially towards the centre (adaxial surface). Multicellular non-glandular and glandular peltate trichomes were present on both the upper and lower epidermis.
The transverse section of the leaf lamina showed the presence of both thickened upper and lower epidermis with cuticle. The upper and lower epidermis cells are big, rectangular, or cuboid-shaped. The lamina is characterized by the presence of single layers of palisade tissue that contain compact cylindrical parenchymal cells with green plastids on the dorsal surface. Spongy tissue comprises of 2-3 layers of spherical parenchyma with intercellular spaces on the ventral surface (Fig. 2D). The lower epidermis has two types of trichomes. The first is nonglandular multicellular covering trichomes. Meanwhile, the second is a glandular peltate consisting of a basal cell, and unicellular stalk and an 8-celled head (2-30 µm in diameter and 20 µm in length) that is a little bit sunken in the epidermis (Fig. 2B & D).

Clerodendrum inerme (L.) Gaertn.
Stem: The transverse section of the stem has a nearly circular outline containing glandular trichomes on the surface. The epidermis of stems consists of a single layer of cells broken at many places due to the pressure of cells produced by phellogen, forming a thin layer of elongated brown, lignified cork cells (Fig. 3A). Secondary cortex is composed of patches of sclerenchyma cells alternating with compressed parenchymatous cells, particularly above the phloem. The secondary phloem is composed of sieve elements and companion cells, which alternate with axial parenchyma cells. Small patches of up to 10 cells or tangential multiples of 4-5 sclerenchyma cells are also present in a ring in the secondary phloem after short intervals of parenchyma cells (Fig. 3C). Secondary phloem rays are uniseriate or 2-3 seriate. Both types of rays are composed mostly of square and upright cells throughout the body (Fig. 3E). Secondary xylem comprises rounded to angular vessels present mostly in radial multiples of 2-3 with some solitary vessels. Vessel diameter is 35 (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) µm and vessel length is 320 (211-403) µm. Perforation plates in vessels are generally simple and rounded, but reticulated perforation plates can be seen in combination with simple perforation plates at a few places (Fig. 3G). Inter-vessel pits are alternate and distinctly bordered. Vessel ray pits are simple to round with a reduced border. The axial parenchyma is scanty paratracheal. Spiral thickening on vessel and fibre elements is also present near the pith region (Fig. 3F). Fibers are thin walled, septate with simple pits on radial walls. Rays are uniseriate to biseriate, composed of square and upright cells only throughout the body. A prismatic and rhomboidal crystal was found in the sclerenchyma cells of secondary phloem (Fig. 4E). A narrow pith made of parenchymatous cells is also present in the centre followed by secondary xylem.

Root:
The transverse section of the root is circular in outline, consisting of 4-6 cells of layered cork followed by cortex and vascular zone (Fig 3B). The cortex is narrow, with stone cells scattered in thin-walled parenchyma. The secondary phloem is thin, containing tangential multiples of 2-3 stone cells after long intervals of parenchyma cells in a ring (Fig. 3D). Stone cells of the cortex as well as secondary phloem containing rhomboidal crystals in its lumens (Fig. 4F). The xylem is well-developed and wider, measuring about three-fourths of the diameter of the cross section. Xylem vessels are rounded to angular in outline, mostly in radial multiples of 2-4 with a few solitary vessels. The vessel diameter is 52 (34-65) µm. Rays are conspicuous, 1-3 seriate. Fibers are thin walled. The axial parenchyma is scanty paratracheal. Pith is absent.
Leaf: The transverse section of the midrib and petiole contain epidermis in a single layer with thickened cuboidal cells. The upper epidermis was followed by collenchyma cells (2-3 layers), and the cortex was formed of several rows of parenchyma cells. Vascular bundle is present in the centre of the midrib. The phloem was composed of parenchyma sieve tubes and thin-walled fibres. Xylem was composed of vessels, wood parenchyma, and thin-walled fibers. The xylem vessels showed spiral thickening. Cortical tissue was made of parenchyma, which occupied its centre. The organization of protoxylem is pointed towards the adaxial surface, while it is on the abaxial surface in the case of metaxylem. Glandular peltate types of trichomes exist on both the upper and lower epidermis (Fig. 4A).
In the cross-section of the leaf lamina, the upper and lower epidermises are made up of big, rectangular, or cuboid-shaped cells thickened with cuticle. The lamina is characterized by the presence of palisade and spongy layers on the dorsal and ventral surfaces, respectively. Below the upper epidermis, single layered compact cylindrical palisade tissue with green plastids is found in the midrib region (Fig. 4B). Spongy tissue comprises spherical parenchyma cells (2-3 layers) containing intercellular spaces. Non-glandular trichomes were very rare, and small (20-30 µm in length) on the upper epidermis only, while glandular trichomes (20 µm in length) are present on the lower epidermis. Glandular trichomes are a little bit sunken in the epidermis (Fig. 4D).

Stem:
The transverse section of the stem revealed a nearly circular outline covered with non-glandular and glandular peltate trichomes (Fig. 6G). The epidermis of stems consists of a single layer cell. In the young stem, the bark is composed of a narrow secondary cortex composed of 4-5 rows of compressed parenchymatous cells. At maturity or after secondary growth, the epidermis broken out and 4-5 cork are formed by phellogen. The thin layer of cork cells is formed of elongated brown cells, or lignified cells. The secondary phloem is composed of tangential zones of sieve elements and companion cells with axial parenchyma cells. Secondary phloem rays may be uniseriate or biseriate. Small patches of 4-5 sclerenchyma cells are also present in the secondary phloem after some intervals in a ring. 1-2 celled layers of vascular cambium are present following secondary phloem (Fig. 5A & C). Secondary xylem comprises rounded to angular vessels that are mostly solitary with a few radial multiples of 2-3. Perforation plates in vessels are simple, rounded to oval, with distinctly bordered alternate pits on their lateral walls (Fig. 5F). The axial parenchyma is scanty paratracheal. Fibers are thin walled, septate, with simple pits on radial walls. Rays are uniseriate to biseriate, composed of square and upright cells only throughout the body (Fig. 5E). Vessel ray pits are simple, rounded to horizontal/gash-like, with a reduced border. Abundant chambered prismatic / rhomboidal crystals are present in secondary phloem fibers (Fig. 6E & F). A well-developed pith of parenchymatous cells is also present in the centre followed by secondary xylem.

Root:
The transverse section of the root is circular in outline, consisting of 4-5 layers of cork cells, followed by the cortex and vascular zone. The secondary phloem is narrow with scattered sclerenchyma fiber patches of 5-6 cells, and the cambium is formed into a ring. The xylem is well-developed and wider, measuring about three-fourths of the diameter of the cross section. Xylem vessels with a rounded to oval outline, mostly solitary with a few radial or tangential multiples of 2 or 3 ( Fig. 5B & D). Xylem fibers are thin to thick walled. Rays are conspicuous, both uniseriate and biseriate. Pith is absent in the mature stage of the root. Abundant chambered prismatic /rhomboidal crystals are present in the secondary phloem fibers (Fig.  6F).

Leaf:
The cross section of the petiole and midrib was convex on the adaxial surface with a single layered epidermis of cuboidal cells. Two or three layers of collenchyma cells were present below the upper epidermis. A collateral vascular bundle present in the centre of the midrib. Xylem and phloem were present in vascular bundles. The arrangement of the protoxylem and metaxylem was similar to that of C. inerme (Fig. 6A & C).
The cross-section of the lamina showed thickening of the cuticle on both the upper and lower epidermis. Both the epidermis cells consist of big, rectangular or cuboidshaped cells. The lamina was characterized by the presence of palisade and spongy layers on the dorsal and ventral surfaces, respectively. A highly compact single layer of cylindrical cells of palisade tissue was present below the upper epidermis. One or two layers of spherical parenchyma cells containing intercellular spaces were present in spongy tissue (Fig. 6B). Both glandular and nonglandular trichomes were present on the epidermis. Non-

Molecular Analysis
The result obtained in the current study also helps in the generation of DNA barcoding for the above-mentioned species, as the markers used are well established for the generation of DNA barcodes especially rbcl, matk, psba-trnH, and ITS (25,26). In molecular analysis, 3 chloroplast markers (matK, rbcl and trnH-psbA) and one nuclear marker (ITS) were used. Though we are unable to amplify matk as we have used the primer taken from a single position, it may be amplified using primers from different positions (loci). The isolated genomic DNA was used to amplify the markers, and single bands were obtained for rbcl, trnH-psbA, and ITS (Fig. 7) while no bands were observed in case of matK which signifies no amplification. PCR amplification and sequencing of bidirectional reads were the highest for ITS (99 %), followed by rbcL (98%), trnH-psbA (96.7%) and matK (0%). The amplified fragments were then purified and sequenced for further analysis, and a tree was constructed (Fig. 8, 9 and 10). The sequence obtained from Sanger sequencing was blasted using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?  Fig. 8. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [36,37]. The tree with the highest log likelihood (-4057.61) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved 5 nucleotide sequences. There were a total of 784 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 PAGE_TYPE=BlastSearch), NCBI and found more than 95% homogeneity. The phylogenetic tree constructed for all the markers (chloroplast and nuclear) depicts a close relationship between C. inerme and P. serratifolia and a distant relationship with C. phlomidis (Fig. 8, 9 and 10).

Discussion
Currently, increasing demand for herbal drugs leads to drug substitution and adulteration. Exponential exploitation of plant resources is being done to fulfil the needs and demands of herbal industries, which leads to a scarcity of plant resources. Thus, it is mandatory to document, authenticate, and validate the plant resources to maintain the quality of herbal products.
Various controversies are associated with the identification and usage of authentic plant species and plant parts in the case of Agnimantha. The existing literature reports different species of Clerodendrum and Premna in this group, but conclusions cannot be drawn for the correct identification. The differentiation between authentic and substitute sources of Agnimantha is still challenging. Phytochemical studies revealed that all three botanical sources possess varying amount of the marker compound verbascoside. Interestingly, the principle active compound was found to be higher in substitute sources such as P. mollissima and P. serratifolia than the authentic source C. phlomidis, leading to the possibility that P. serratifolia and P. mollissima could be the potential sources of Agnimantha (27).
There were a few molecular studies (28-31) already conducted on the species under study, but no reference was found regarding the relationship among these species. Similar studies on different varieties like Safed zeera adulteration in Kala zeera has been detected using molecular markers i.e., ITS2 and psbA-trnH. The amplified product of psbA-trnH barcode was different in Safed zeera (322 bp) as compared to kala zeera (257 bp) which can be easily resolved on agarose gel (32). However, morphological and anatomical studies reflect the relationship between these species on the basis of their external and structural features, but there is a lacuna in molecular based information. The current molecular phylogenetic and anatomical study involving the abovementioned species plays a critical role in determining the authenticity of the source plant in the ayurvedic drug Agnimantha.
Anatomical characterization of the species involved revealed that all three species under the name Agnimantha are considerably similar in the anatomy of the root, stem, and leaves, except for some features. Though the species under study are closely related to each other, on the basis of differential anatomical features (Table 6) of these Agnimantha species, we can conclude that C. inerme is more closely related to P. serratifolia than to C. phlomidis. Moreover, the results of molecular analysis also corroborate the results obtained from anatomical studies  Fig. 9. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [36,37]. The tree with the highest log likelihood (-4596.40) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved 5 nucleotide sequences. There were a total of 784 positions in the final dataset. Evolutionary analyses were conducted in MEGA11

trnH -psbA
Outgroup Fig. 10. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [36,37]. The tree with the highest log likelihood (-2628.22) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved 5 nucleotide sequences. There were a total of 784 positions in the final dataset. Evolutionary analyses were conducted in MEGA11. and suggest a close relationship between P. serratifolia and C. inerme and a distant relationship with C. phlomidis. Clerodendrum phlomidis is considered the authentic source of Agnimantha, while P. serratifolia and C. inerme are considered substitute sources as they possess equal morphological, anatomical, and phytochemical similarities with authentic species.
The results obtained in the present study are corroborated with the findings of API, but these are not enough to consider C. inerme and P. serratifolia as substitutes. Further Phytochemical and metabolomic based studies need to be conducted to authenticate the main source and substitutes.