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ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the plant Morinda longissima Y.Z.Ruan (Rubiaceae) is used by ethnic people in Vietnam for the treatment of liver diseases and hepatitis. AIM OF THE STUDY: The study was designed to assess the efficacy of the 95% ethanolic extract of Morinda longissima roots (MLE) in experimental immune inflammation. The phytochemical variation of root extract and the chemical structures of natural compounds were also investigated using HPLC-DAD-HR-MS analysis. MATERIALS AND METHODS: Three different doses (100, 200, and 300 mg/kg b.w.) of MLE were chosen to determine anti-inflammatory activity. The mice were given orally extracts and monitored their behavior and mortality for 14 days to evaluate acute toxicity. The volume of the paw and the histopathological evaluation were carried out. The polyphenolic phytoconstituents of MLE extract were identified using LC/MS analysis. The anti-inflammatory efficacy in silico and molecular docking simulations of these natural products were evaluated based on their cyclooxygenase (COX)-1 and 2 inhibitory effects. RESULTS: This investigation showed the 95% ethanolic extract of Morinda longissima roots was found non-toxic up to 2000 mg/kg dose level in an acute study, neither showed mortality nor treatment-related signs of toxicity in mice. Eight anthraquinones and anthraquinone glycosides of Morinda longissima roots were identified by HPLC-DAD-HR-MS analysis. In the in vivo experiments, MLE was found to possess powerful anti-inflammatory activities in comparison with diclofenac sodium. The highest anti-inflammatory activity of MLE in mice was observed at a dose of 300 mg/kg body weight. The in silico analysis showed that seven out the eight anthraquinones and anthraquinone glycosides possess a selectivity index RCOX-2/COX-1 lower than 1, indicating that these compounds are selective against the COX-2 enzyme in the following the order: rubiadin-3-methyl ether < morindone morindone-6-methyl ether < morindone-5-methyl ether < damnacanthol < rubiadin < damnacanthol-3-O-ß-primeveroside. The natural compounds with the best selectivity against the COX-2 enzyme are quercetin (9), rubiadin-3-methyl ether (7), and morindone (4), with RCOX2/COX1 ratios of 0.02, 0.03, and 0.19, respectively. When combined with the COX-2 protein in the MD research, quercetin and rubiadin-3-methyl ether greatly stabilized the backbone proteins and ligands. CONCLUSION: In conclusion, the anthraquinones and ethanolic extract of Morinda longissima roots may help fight COX-2 inflammation. To develop novel treatments for inflammatory disorders linked to this one, these chemicals should be investigated more in the future.

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Callus suspension techniques have been considered attractive for improving bioactive metabolite productivity; methyl jasmonate (MeJA) is a widely used elicitor for stimulating synthetic pathways. In this study, a multivariate analysis-based metabolomics approach was employed to investigate the primary and specialized metabolites in the leaves, unelicited calli, and 100 or 200 µM MeJA elicited calli of Damnacanthus major. Rubiadin, a powerful anthraquinone with various therapeutic properties, was only identified in D. major calli, accumulating in a MeJA elicitation concentration-dependent manner. Callus cultures also contained high levels of amino acids, sugars, and phenolic compounds, indicating energy metabolism and metabolic adaptation responses for proliferation and stabilization. Regarding MeJA application, elicited calli contained higher amounts of quinic acid, kaempferol, and glucose with lower amounts of sucrose and raffinose than those in the unelicited control, which were closely related to protective mechanisms against MeJA. Moreover, excessive elicitation increased the asparagine, fructose, and raffinose levels and decreased the glucose and sucrose levels, which was ascribed to increased activation of the aminoacyl-tRNA biosynthesis pathway and wider utilization of glucose than of fructose after sucrose degradation. These results will be useful for optimizing plant cell culture techniques to achieve high production rates for valuable specialized metabolites.

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Rubiadin (Rub) is a genotoxic component of madder color (MC) that is extracted from the root of Rubia tinctorum L. MC induces renal tumors and preneoplastic lesions that are found in the proximal tubule of the outer stripe of the outer medulla (OSOM), suggesting that the renal carcinogenicity of MC is site specific. To clarify the involvement of Rub in renal carcinogenesis of MC, we examined the distribution of Rub in the kidney of male gpt delta rats that were treated with Rub for 28 days. We used desorption electrospray ionization quadrupole time-of-flight mass spectrometry imaging (DESI-Q-TOF-MSI), along with the histopathological analysis, immunohistochemical staining, and reporter gene mutation assays of the kidney. DESI-Q-TOF-MSI revealed that Rub and its metabolites, lucidin and Rub-sulfation, were specifically distributed in the OSOM. Histopathologically, karyomegaly characterized by enlarged nuclear and microvesicular vacuolar degeneration occurred in proximal tubule epithelial cells in the OSOM. The ɤ-H2AX- and p21-positive cells were also found in the OSOM rather than the cortex. Although dose-dependent increases in gpt and Spi- mutant frequencies were observed in both the medulla and cortex, the mutant frequencies in the medulla were significantly higher. The mutation spectra of gpt mutants showed that A:T-T:A transversion was predominant in Rub-induced gene mutations, consistent with those of MC. Overall, the data showed that the distribution of Rub and its metabolites resulted in site-specific histopathological changes, DNA damage, and gene mutations, suggesting that the distribution of genotoxic components and metabolites is responsible for the site-specific renal carcinogenesis of MC.

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As an active substance isolated from the root of Morinda officinalis How., rubiadin-1-methyl ether (RBM), can improve osteoporosis due to its inhibition on osteoclastogenesis. Autophagy plays a key role in osteoclastogenesis. Our research aims to explore the relationship between RBM, autophagy, and osteoclastogenesis. Our results showed that RBM not only inhibited the differentiation level of osteoclasts and the proliferation ability of osteoclast precursors (OCPs), but also repressed the autophagic activity in OCPs (LC3 transformation and the number of autophagosomes observed by transmission electron microscopy). However, RBM-inhibited osteoclast differentiation and OCP autophagy (LC3 transformation and LC3-puncta formation) could be reversed by the application of TAT-Beclin1. Moreover, RBM administration reduced RANKL-induced p65 phosphorylation and p65 nuclear translocation in OCPs. In addition, the addition of RBM inhibited Beclin1 protein level and BECN1 (the gene form of Beclin1) mRNA level in OCPs increased by RANKL. Importantly, the reduction in the expression of BECN1 and Beclin1, LC3 transformation, and osteoclastic differentiation in OCPs caused by RBM were reversed by p65 overexpression. In conclusion, RBM may reduce the transcription of BECN1 by inhibiting the activation of nuclear factor kappa B (NF-κB) p65, thereby inhibiting Beclin1-dependent autophagy and RANKL-induced osteoclastogenesis.

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Rubiadin is identified as a bioactive anthraquinone that exists in some quinone rich plants. The current research was carried out to evaluate the potential anti-inflammatory impact of Rubiadin in acute and chronic inflammation test models in rodents. The anti-inflammatory activity of Rubiadin was examined in cotton pellet-induced granuloma and carrageenan-induced edema as chronic and acute inflammation models in rats. TNF-α level and histopathological changes were assessed using sampled foot tissue of rat in the acute model. Also, the IL-1β level was assessed in the chronic model. One-way ANOVA (post hoc Tukey’s) analysis was used for comparing the groups. Rubiadin (0.5 mg/kg, i.p.) induced a significant reduction in TNF α level and the paw edema compared to the control group in carrageenan test. Also, it was observed that the anti-inflammatory activity of Rubiadin (0.5 mg/kg, i.p.) is comparable to mefenamic acid (30 mg/kg, i.p.) as the standard drug. Rubiadin was effective in granuloma induced by cotton pellet concerning the granuloma and transudate formation amount. Rubiadin's anti-inflammatory effects were associated with a significant IL-1β decrease in this model. The results suggest that Rubiadin as a natural compound can possess significant peripheral anti-inflammatory impacts.

A rubiadina é identificada como uma antraquinona bioativa que existe em algumas plantas ricas em quinonas. A presente pesquisa foi realizada para avaliar o potencial impacto anti-inflamatório da rubiadina em modelos de teste de inflamação aguda e crônica em roedores. A atividade anti-inflamatória da rubiadina foi examinada em granuloma induzido por pellet de algodão e edema induzido por carragenina como modelos de inflamação crônica e aguda em ratos. O nível de TNF-α e as alterações histopatológicas foram avaliados usando amostra de tecido do pé de rato no modelo agudo. Além disso, o nível de IL-1β foi avaliado no modelo crônico. A análise ANOVA de uma via (post hoc de Tukey) foi usada para comparar os grupos. A rubiadina (0,5 mg / kg, i.p.) induziu uma redução significativa no nível de TNF α e no edema da pata em comparação com o grupo de controle no teste de carragenina. Além disso, foi observado que a atividade anti-inflamatória da rubiadina (0,5 mg / kg, i.p.) é comparável ao ácido mefenâmico (30 mg/kg, i.p.) como o fármaco padrão. A rubiadina foi eficaz no granuloma induzido por pellet de algodão no que diz respeito à quantidade de granuloma e formação de transudato. Os efeitos anti-inflamatórios da rubiadina foram associados a uma redução significativa de IL-1β nesse modelo. Os resultados sugerem que a rubiadina como um composto natural pode ter impactos anti-inflamatórios periféricos significativos.

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Abstract: Rubiadin is identified as a bioactive anthraquinone that exists in some quinone rich plants. The current research was carried out to evaluate the potential anti-inflammatory impact of Rubiadin in acute and chronic inflammation test models in rodents. The anti-inflammatory activity of Rubiadin was examined in cotton pellet-induced granuloma and carrageenan-induced edema as chronic and acute inflammation models in rats. TNF- level and histopathological changes were assessed using sampled foot tissue of rat in the acute model. Also, the IL-1 level was assessed in the chronic model. One-way ANOVA (post hoc Tukeys) analysis was used for comparing the groups. Rubiadin (0.5 mg/kg, i.p.) induced a significant reduction in TNF level and the paw edema compared to the control group in carrageenan test. Also, it was observed that the anti-inflammatory activity of Rubiadin (0.5 mg/kg, i.p.) is comparable to mefenamic acid (30 mg/kg, i.p.) as the standard drug. Rubiadin was effective in granuloma induced by cotton pellet concerning the granuloma and transudate formation amount. Rubiadins anti-inflammatory effects were associated with a significant IL-1 decrease in this model. The results suggest that Rubiadin as a natural compound can possess significant peripheral anti-inflammatory impacts.

Resumo A rubiadina é identificada como uma antraquinona bioativa que existe em algumas plantas ricas em quinonas. A presente pesquisa foi realizada para avaliar o potencial impacto anti-inflamatório da rubiadina em modelos de teste de inflamação aguda e crônica em roedores. A atividade anti-inflamatória da rubiadina foi examinada em granuloma induzido por pellet de algodão e edema induzido por carragenina como modelos de inflamação crônica e aguda em ratos. O nível de TNF- e as alterações histopatológicas foram avaliados usando amostra de tecido do pé de rato no modelo agudo. Além disso, o nível de IL-1 foi avaliado no modelo crônico. A análise ANOVA de uma via (post hoc de Tukey) foi usada para comparar os grupos. A rubiadina (0,5 mg / kg, i.p.) induziu uma redução significativa no nível de TNF e no edema da pata em comparação com o grupo de controle no teste de carragenina. Além disso, foi observado que a atividade anti-inflamatória da rubiadina (0,5 mg / kg, i.p.) é comparável ao ácido mefenâmico (30 mg/kg, i.p.) como o fármaco padrão. A rubiadina foi eficaz no granuloma induzido por pellet de algodão no que diz respeito à quantidade de granuloma e formação de transudato. Os efeitos anti-inflamatórios da rubiadina foram associados a uma redução significativa de IL-1 nesse modelo. Os resultados sugerem que a rubiadina como um composto natural pode ter impactos anti-inflamatórios periféricos significativos.

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Madder color (MC), a natural dye isolated from Rubia tinctorum, is a potent carcinogen that targets the outer stripe of outer medulla (OSOM) in the kidneys of rats. To clarify the role of MC components in renal carcinogenesis, we examined distributions of MC components and metabolites in the kidneys of rats treated with MC using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Alizarin, lucidin, munjistin, nordamnacanthal, purpurin, pseudopurpurin, rubiadin, and some other metabolites detected and identified by liquid chromatography time-of-flight MS analysis of rat serum 1 h after MC administration were subjected to DESI-MSI. This analysis enabled visualization of the distribution of anthraquinones in the kidney, and the ion images showed a characteristic distribution according to their chemical structure. Among the components, lucidin and rubiadin specifically localized in the OSOM, suggesting that their genotoxicity was a direct cause of MC carcinogenesis. Alizarin showed greater distribution in the OSOM than the cortex and may therefore participate in renal carcinogenicity owing to its tumor-promoting activity. Overall, our data suggested that the distribution of carcinogenic components to the OSOM was responsible for the site-specific renal carcinogenicity of MC and that DESI-MSI analysis may be a powerful tool for exploring the mechanisms of chemical carcinogenesis.

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Diabetic nephropathy (DN) is a significant source of end-stage renal illness all over the world in both developed and developing countries. The aim of the study was to optimize rubiadin-loaded niosomes (RLN) using Box-Behnken design for the management of streptozotocin-nicotinamide (STZ-NA)-induced DN in Wistar rats. The RLN were formulated by a "thin-layer hydration technique." The optimization of RLN was done by Box-Behnken design; the independent variables were cholesterol (CHOL), Span 80, and methanol, while the dependent factors were the vesicle size, zeta potential, and entrapment efficiency. The optimized formulation was characterized for various biochemical parameters including anti-diabetic activity in Wistar rats. The optimized RLN presented vesicle size of 238 nm, zeta potential -68 mV, and entrapment efficiency 85%. A noteworthy decreased in blood glucose level was detected in STZ-NA-induced DN rats when orally treated with RLN (100 mg/kg/week and 200 mg/kg/week). Oral administration of RLN formulation considerably decreased the levels of urea, uric acid, and creatinine in DN rats. In addition, treatment of DN rats with RLN formulation considerably improves the level of TBARS, GSH, SOD, and CAT. The lipid profile of DN rats was also improved on treatment with RLN formulation. This study revealed that the prepared RLN formulation was successfully optimized by Box-Behnken design and found to be useful for the management of STZ-NA-induced DN in Wistar rats.

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Anthraquinones (AQs) are found in a variety of consumer products, including foods, nutritional supplements, drugs, and traditional medicines, and have a wide range of pharmacological actions. Rubiadin, a 1,3-dihydroxy-2-methyl anthraquinone, primarily originates from Rubia cordifolia Linn (Rubiaceae). It was first discovered in 1981 and has been reported for many biological activities. However, no review has been reported so far to create awareness about this molecule and its role in future drug discovery. Therefore, the present review aimed to provide comprehensive evidence of Rubiadin's phytochemistry, biosynthesis, physicochemical properties, biological properties and therapeutic potential. Relevant literature was gathered from numerous scientific databases including PubMed, ScienceDirect, Scopus and Google Scholar between 1981 and up-to-date. The distribution of Rubiadin in numerous medicinal plants, as well as its method of isolation, synthesis, characterisation, physiochemical properties and possible biosynthesis pathways, was extensively covered in this review. Following a rigorous screening and tabulating, a thorough description of Rubiadin's biological properties was gathered, which were based on scientific evidences. Rubiadin fits all five of Lipinski's rule for drug-likeness properties. Then, the in depth physiochemical characteristics of Rubiadin were investigated. The simple technique for Rubiadin's isolation from R. cordifolia and the procedure of synthesis was described. Rubiadin is also biosynthesized via the polyketide and chorismate/o-succinylbenzoic acid pathways. Rubiadin is a powerful molecule with anticancer, antiosteoporotic, hepatoprotective, neuroprotective, anti-inflammatory, antidiabetic, antioxidant, antibacterial, antimalarial, antifungal, and antiviral properties. The mechanism of action for the majority of the pharmacological actions reported, however, is unknown. In addition to this review, an in silico molecular docking study was performed against proteins with PDB IDs: 3AOX, 6OLX, 6OSP, and 6SDC to support the anticancer properties of Rubiadin. The toxicity profile, pharmacokinetics and possible structural modifications were also described. Rubiadin was also proven to have the highest binding affinity to the targeted proteins in an in silico study; thus, we believe it may be a potential anticancer molecule. In order to present Rubiadin as a novel candidate for future therapeutic development, advanced studies on preclinical, clinical trials, bioavailability, permeability and administration of safe doses are necessary.

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The long history of madder as a source of red dyes and pigments is presented. The variety of plant sources and the range of anthraquinone components discovered over a long period are addressed. Topics such as analysis, industrial uses, biological staining, red bone staining in live animals and toxicity are outlined briefly. The contributions of many chemists are acknowledged.

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Rubiadin-1-methyl ether (RBM) is a natural anthraquinone compound isolated from the root of Morinda officinalis How. In our previous study, RBM was found to have inhibitory effects on the TRAP activity of osteoclasts, which means that RBM may be a candidate for therapy of bone diseases characterized by enhanced bone resorption. However, the further effect of RBM on osteoclasts and the underlying mechanism remain unclear. In the present study, we investigated the effects of RBM isolated from Morinda officinalis How. on osteoclasts derived from bone marrow macrophages (BMMs) and the underlying mechanism in vitro. RBM at the dose that did not affect the viability of cells significantly inhibited RANKL-induced osteoclastogenesis and actin ring formation of osteoclast, while RBM performed a stronger effect at the early stage. In addition, RBM downregulated the expression of osteoclast-related proteins, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cellular oncogene Fos (c-Fos), matrix metallopeptidase 9 (MMP-9) and cathepsin K (CtsK) as shown by Western blot. Furthermore, RBM inhibited the phosphorylation of NF-κB p65 and the degradation of IκBα as well as decreased the nuclear translocation of p65. Collectively, the results suggest that RBM inhibit osteoclastic bone resorption through blocking NF-κB pathway and may be a promising agent for the prevention and treatment of bone diseases characterized by excessive bone resorption.

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ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plant Morinda officinalisHow. (MO) and its root have long been used in traditional medicines in China and northeast Asia as tonics for nourishing the kidney, strengthening the bone and enhancing immunofunction in the treatment of impotence, osteoporosis, depression and inflammatory diseases such as rheumatoid arthritis and dermatitis. AIM OF THE REVIEW: This review aims to sum up updated and comprehensive information about traditional usage, phytochemistry, pharmacology and toxicology of MO and provide insights into potential opportunities for future research and development of this plant. METHODS: A bibliographic investigation was performed by analyzing the information available on MO in the internationally accepted scientific databases including Pubmed, Scopus and Web of Science, SciFinder, Google Scholar, Yahoo, Ph.D. and M.Sc. dissertations in Chinese. Information was also obtained from some local and foreign books on ethnobotany and ethnomedicines. RESULTS: The literature supported the ethnomedicinal uses of MO as recorded in China for various purposes. The ethnomedical uses of MO have been recorded in many regions of China. More than 100 chemical compounds have been isolated from this plant, and the major constituents have been found to be polysaccharides, oligosaccharides, anthraquinones and iridoid glycosides. Crude extracts and pure compounds of this plant are used as effective agents in the treatment of depression, osteoporosis, fatigue, rheumatoid arthritis, and infertility due to their anti-depressant, anti-osteoporosis, pro-fertility, anti-radiation, anti-Alzheimer disease, anti-rheumatoid, anti-fatigue, anti-aging, cardiovascularprotective, anti-oxidation, immune-regulatory, and anti-inflammatory activities. Pharmacokinetic studies have demonstrated that the main components of MO including monotropein and deacetyl asperulosidic acid are distributed in various organs and tissues. The investigation on acute toxicity and genotoxicity indicated that MO is nontoxic. There have no reports on significant adverse effect at a normal dose in clinical application, but MO at dose of more than 1000mg/kg may cause irritability, insomnia and unpleasant sensations in individual cases. CONCLUSION: MO has emerged as a good source of traditional medicines. Some uses of this plant in traditional medicines have been validated by pharmacological investigations. However, the molecular mechanism, structure-activity relationship, and potential synergistic and antagonistic effects of its multi-components such as polysaccharides, oligosaccharides, anthraquinones and iridoid glycosides need to be further elucidated, and the structural feature of polysaccharides also need to be further clarified. Sophisticated analytical technologies should be developed to comprehensively evaluate the quality of MO based on HPLC-fingerprint and content determination of the active constituents, knowing that these investigations will help further utilize this plant.

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Prismatomeris connata was a kind of Rubiaceae plant for treatment of hepatitis, hepatic fibrosis and silicosis. Whereas, the effective components of Prismatomeris connata remains unexplored. The aim of this study was to investigate the inhibitory effects and mechanisms of Rubiadin isolated from Prismatomeris connata against HBV using HepG2.2.15 cells. The levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay. HBV DNA was qualified q-PCR. Rubiadin was isolated by silica gel column. The structure of the compound was elucidated by HPLC, FT-IR, 1 H-NMR, 13 C-NMR and identified as 1,3-Dihydroxy-2-methyl-9, 10-anthraquinone. Rubiadin significantly decreased HBeAg,HBcAg secretion level and inhibit HBV DNA replication. Rubiadin inhibits the proliferation of the cells and HBx protein expression in a dose-dependent manner. The intracellular calcium concentration was significantly reduced. These results demonstrated that Rubiadin could inhibit HepG2.2.15 cells proliferation, reduce the level of HBx expression, and intracellular free calcium, which might become a novel anti-HBV drug candidate.

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ETHNOPHARMACOLOGICAL RELEVANCE: As a classical prescription of traditional Chinese medicine (TCM), Jia-Jian-Di-Huang-Yin-Zi decoction (JJDHYZ) has been used to treat the symptoms of neurological disorders with a long history. AIM OF THE STUDY: To evaluate the effects and possible mechanisms of JJDHYZ on a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced subacute mouse model of Parkinson's disease. MATERIALS AND METHODS: Adult male C57BL/6 mice were randomly divided into five groups: control, MPTP, JJDHYZ low dosage (JJDHYZ-L, 8.5g/kg/day), medium dosage (JJDHYZ-M, 17g/kg/day) and high dosage (JJDHYZ-H, 34g/kg/day). Behavioral tests, immunohistochemistry, immunofluorescence, and high-performance liquid chromatography (HPLC) were conducted to evaluate the neuroprotective effects of JJDHYZ. The mechanism was further explored using TdT-mediated dUTP nick end labeling staining and transmission electron microscopy. The protein expression of Bax, Bcl-2, cytochrome c, full-length caspase9, cleaved caspase9, cleaved caspase3, caspase12 and C/EBP homologous protein was assessed. The toxicity on hepatocytes and renal cells was detected using the enzyme-linked immunosorbent assay kits. RESULTS: JJDHYZ-H restored the behavior performance impaired by MPTP, and reduced the loss of tyrosine hydroxylase. Additionally, it blocked the apoptosis, activated cleaved caspase3 and protected the ultrastructural integrity of mitochondria by regulating the expression of proteins in both mitochondrial and endoplasmic reticulum (ER) caspase12 pathways. CONCLUSIONS: JJDHYZ-H showed behavior recovery and dopamine neuron protection by inhibiting the apoptotic activities associated with mitochondrial and ER caspase12 pathways.

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Manjisthadi churna has been traditionally used in the Ayurvedic system of medicine and by traditional medical practices of India to treat hyperlipidemia. A rapid, simple and accurate method with high performance thin layer chromatography has been developed to standardised Manjisthadi churna using rubiadin, sennoside and ellagic acid as markers. Methanol extract of Manjisthadi churna were used for high performance thin layer chromatography on silica gel plates. The Rf of rubiadin, sennoside-A and ellagic acid were found to 0.48, 0.23 and 0.72, respectively with densitometric scanning at 280 nm and the calibration plot were linear in the range of 100-600 ng of markers. The correlation coefficients were higher than 0.99 were indicative of good linear dependence of peaks area on concentration. The rubiadin, sennoside-A and ellagic acid contents in Manjisthadi churna were found to be 0.014, 0.038 and 0.534% w/w, respectively. This method permits reliable quantification of rubiadin, sennoside-A and ellagic acid with good resolution and separation of the same from other constitutes of the extract of Manjisthadi churna. Recovery value from 95.66-102.33% showed the reliability and reproducibility of the method. The proposed high performance thin layer chromatography method for simultaneous quantification of markers in Manjisthadi churna can be used for routine quality testing.