Rhinoceros horn mineral and metal concentrations vary by sample location, depth, and color.

Poaching is again driving rhinos to the brink of extinction due to the demand for rhino horn products consumed for cultural, medicinal, and social purposes. Paradoxically, the same horn for which rhinos are killed may contain valuable clues about the species' health. Analyses of horn composition could reveal such useful bioindicators while elucidating what people actually ingest when they consume horn derivatives. Our goals were to quantify minerals (including metals) in rhino horn and investigate sampling factors potentially impacting results. Horns (n = 22) obtained during necropsies of white (n = 3) and black (n = 13) zoo rhinos were sampled in several locations yielding 182 specimens for analysis. Initial data exposed environmental (soil) contamination in the horn's exterior layer, but also confirmed that deep (≥ 1 cm), contaminant-free samples contained measurable concentrations of numerous minerals (n = 18). Of the factors examined in deep samples, color-associated mineral differences were the most profound with dark samples higher in zinc, copper, lead, and barium (p < 0.05). Our data demonstrate that rhino horns contain both essential and potentially toxic minerals that could be relevant to rhino health status, but low concentrations make their human health benefits or risks unlikely following consumption.

[Identification of horn-derived animal medicines based on detection of keratin-derived characteristic peptides].

Due to the highly stable structure of keratin, the extraction and dissolution steps of animal medicines rich in keratin are complex, which seriously restricts the detection efficiency and flux. Therefore, this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-derived animal medicines. For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA), the ion pair conditions of the characteristic peptides were optimized, and the retention time, intensity and other data of the specific peptides were compared between the samples treated with/without IAA. Two pre-treatment methods, direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis, were used to prepare horn-derived animal medicine samples. The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn, water buffalo horn, goat horn, and yak horn were compared regarding the retention time of specific peptides and ion intensity. The results indicated that after direct enzymatic hydrolysis, the specific peptides in the samples without IAA treatment can be detected. Compared with the characteristic peptides in the samples treated with IAA, their retention time shifted back and the mass spectrometry response slightly decreased. The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines. Overall, the process of direct enzymatic hydrolysis can be used to treat horn samples, omitting the steps of protein extraction and dithiothreitol and IAA treatment, significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines. This study provides ideas for quality research and standard improvement of horn-derived animal medicines.

Combination of mathematics and label-free proteomics for discovering keratin-derived specific peptide biomarkers to distinguish animal horn-derived traditional Chinese medicines.

Saiga antelope horn and Rhinoceros horn have been used in traditional Chinese medicine for thousands of years. However, due to the protection of wildlife, the application of these rare animal horns has been restricted or prohibited. Therefore, water buffalo horn, goat horn, and yak horn have been applied as alternatives to Rhinoceros horn or Saiga antelope horn in a clinic. It is extremely difficult to distinguish normal animal horns in powdered or decocted form, especially identifying related species such as water buffalo horn, yak horn, and cattle horn. In this work, mathematics set and label-free proteomics analysis were combined for discovering keratin-derived specific peptide biomarkers. By using mathematics set analysis after nano liquid chromatography-tandem mass spectrometry-based proteomics, the selected species-specific peptides could be used to identify the authenticity of the Saiga antelope horn and goat horn. Furthermore, peptide biomarkers were selected to distinguish related species-derived horns, water buffalo horn, yak horn, and cattle horn. In total, eight peptide biomarkers were selected and applied for simultaneously distinguishing different horn samples. The present strategy provides a method for peptide biomarkers discovery and also has positive significance for ensuring the quality and efficacy of animal horn-derived traditional Chinese medicines and their products.

[Identification of specific peptides from Bubali Cornu by ultra-performance liquid chromatography-tandem mass spectrometry].

This paper explored the specific peptides from Bubali Cornu by ultra-performance liquid chromatography-tandem mass spectrometry and based on mathematics set theory. Following the profile analysis of peptides from Bubali Cornu, Bovis Grunniens Cornu, Caprae Hircus Cornu, and Suis Cornu by nano LC-LTQ-Obitrap-MS after digestion with trypsin, the relationship of peptide composition among different samples was analyzed using the mathematics set theory. The ones that existed only in the Bubali Cornu set rather than in any other set were considered as the specific peptides of Bubali Cornu. The further bioinformatic analysis revealed four specific peptides from Bubali Cornu, whose specificity was verified by UPLC-QQQ-MS. The results showed that these four peptides could be used for distinguishing Bubali Cornu from Caprae Hircus Cornu and Suis Cornu. This study has provided a rapid and simple method for seeking the specific peptides in animal medicines, which can be utilized for quality evaluation of animal medicines, thus making them authenticable and traceable.

Lead uptake into calcified and keratinized compartments of horns from a convenience sample of lead-dosed goats.

Hair and/or nail analyses are sometimes used in biomonitoring studies due to the convenience of sample collection, storage, and transport, as well as the potential to assess past exposures to toxic metals, such as lead (Pb). However, the validity of Pb measurements in these keratinized matrices as biomarkers of absorbed dose remains unclear. The aim of this study was to examine the uptake of Pb into horns harvested postmortem from 11 goats that received a cumulative oral dose of up to 151 g Pb acetate over a period of 1-11 years as part of a long-term blood Pb proficiency testing program. Uptake of Pb into keratinized horn was compared to the corresponding underlying bony horn core, which, as part of the bone compartment, provided a measure of absorbed Pb dose. Two complementary analytical techniques were used to assess Pb: X-Ray Fluorescence (XRF) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Detectable amounts of Pb were found in all keratinized horn samples (0.45-6.6 µg/g) and in all but one bony core sample (1.4-68 µg/g). In both bony core and keratinized horn samples, Pb accumulation increased with dose over a low-to-moderate cumulative-dose interval, consistent with previous observations, but plateaued at higher doses. Significant associations were observed between Pb in keratinized horn and bony core samples particularly with XRF measurements, which represent the surface bone compartment. These findings provide evidence that Pb is excreted in keratinized tissues but reflects only a small fraction of the absorbed Pb dose, likely transferred from underlying bone tissue.

Development of an on-line Raman spectral analytical method for monitoring and endpoint determination of the Cornu Caprae Hircus hydrolysis process.

OBJECTIVES: Cornu Caprae Hircus (goat horn, GH), a medicinal animal horn, is frequently used in traditional Chinese medicine, and hydrolysis is one of the most important processes for GH pretreatment in pharmaceutical manufacturing. In this study, on-line Raman spectroscopy was applied to monitor the GH hydrolysis process by the development of partial least squares (PLS) calibration models for different groups of amino acids. METHODS: Three steps were considered in model development. In the first step, design of experiments (DOE)-based preprocessing method selection was conducted. In the second step, the optimal spectral co-addition number was determined. In the third step, sample selection or reconstruction methods based on hierarchical clustering analysis (HCA) were used to extract or reconstruct representative calibration sets from the pool of hydrolysis process samples and investigated for their ability to improve model performance. KEY FINDINGS: This study has shown the feasibility of using on-line Raman spectral analysis for monitoring the GH hydrolysis process based on the designed measurement system and appropriate model development steps. CONCLUSIONS: The proposed Raman-based calibration models are expected to be used in GH hydrolysis process monitoring, leading to more rapid material information acquisition, deeper process understanding, more accurate endpoint determination and thus better product quality consistency.

Creating artificial Rhino Horns from Horse Hair.

Demand for rhino horn is driving poaching with devastating effect for the few individuals left of the few species surviving from this once numerous, widespread and cosmopolitan clade of pachyderms. We bundled together tail hairs of the rhino's ubiquitous near relative, the horse, to be glued together with a bespoke matrix of regenerated silk mimicking the collagenous component of the real horn. This approach allowed us to fabricate composite structures that were confusingly similar to real rhino horn in look, feel and properties. Spectral and thermal FT-IR, DSC and TGA analysis demonstrated the similar chemical composition and thermo-mechanical properties between the natural and the faux horns.

Trace element concentrations in horn: Endogenous levels in keratin and susceptibility to exogenous contamination.

As a recorder containing both physiological and environmental information, keratinized tissues, such as hair and feather, can be used to reveal geographical information, to monitor the exposure to pollutants, and to reconstruct dietary history. However, trace element analysis of keratinized tissues is complicated by the lack of reference endogenous ranges of trace element and the lack of understanding of the susceptibility of each element to exogenous contamination. The interior of animal horn is the cleanest of all keratinized tissues with minimum exogenous contamination because of its large size. Thus, the trace element concentrations in horn interior samples can provide reliable endogenous concentration ranges. Here we measured the concentrations of trace elements in horn interior samples of cattle and wild animals, which we propose to be used as the reference ranges for endogenous levels of trace elements in keratin. We calculated the enrichment factors of 30 trace elements in horn interior samples relative to the continental crust, which we considered the average exogenous contamination. We compared the ranges of elemental concentrations measured in horn interior samples, in the order of decreasing enrichment factor, to their reference ranges in hair, fingernails, and toenails, as well as their concentrations in caprine horns. Such comparison validates the use of the enrichment factor as an indicator of the susceptibility of an element to contamination: an element with a high enrichment factor is generally less likely to be affected by contamination and vice versa.

A study of lead uptake and distribution in horns from lead-dosed goats using synchrotron radiation-induced micro X-ray fluorescence elemental imaging.

OBJECTIVE: The principal goal of this study was to investigate the uptake and distribution of lead (Pb) in the horns of Pb-dosed goats, and to explore possible links to their historical Pb dosing records. Horn is a keratinized material that grows in discrete increments with the potential to preserve the historical record of past environmental exposures. While previous studies have leveraged this potential to examine environmental and biological phenomena in horns, Pb uptake has never been explored. METHODS: Horns were collected post-mortem from three goats that had been previously used to produce blood lead reference materials for the New York State proficiency testing program. The animals were periodically dosed with lead acetate, administered orally in a capsule, over a 5 to 8-year period. Horn cross sections were taken from each animal and analyzed using synchrotron radiation-induced micro X-ray fluorescence spectrometry (SR-µXRF) at the Cornell High Energy Synchrotron Source (CHESS). RESULTS: Elemental distribution maps were obtained by SR-µXRF for Pb, Ca, S, Se, and three other elements (Br, Zn and Cu), with values reported quantitatively as a mass fraction (µg/g for trace elements and mg/g for Ca and S). Accumulations of Pb were clearly visible as a series of narrow "rings" in each of the horn samples analyzed. The elements Ca, S, Br, Zn, and Cu were also detected as discrete rings within each cross-section, with Br strongly correlated with S in the samples examined. A marginal increase in Se may coincide with Pb accumulation in horn cross-sections. Annual mineralization estimates based on the relative distribution of Ca and S were used to establish a tentative timeline for horn growth, with each timeline linked to the pattern of Pb accumulation in the corresponding horn cross-section sample. CONCLUSIONS: Following ingestion, absorbed Pb is eventually deposited into caprine horns, resulting in discrete accumulations or "rings." Elemental mapping by SR-µXRF clearly show Ca-rich layers that vary with annual periodicity, consistent with previous reports of horn mineralization. Localized enrichment of Cu, Zn, Br and S appear to coincide with the keratinized regions related to the annual growth ring pattern in horns. Spatial analysis of horns for Pb accumulation may be useful as a qualitative marker of time-resolved exposures that may reflect specific periods of acute Pb absorption.

Comparison of the peptidome released from keratins in Saiga antelope horn and goat horn under simulated gastrointestinal digestion.

Goat horn (Caprae Hircus Cornu, GH) has been used as a substitute for Saiga antelope horn (Saigae Tataricae Cornu, SAH) in the clinic and the pharmaceutical industry. In the present study, peptides released from SAH and GH under simulated gastric and intestinal digestion were identified. The results showed that most of the peptides released from SAH and GH under simulated gastrointestinal digestion were hydrophilic, and over 75% of the peptides from keratins (KRTs) were hydrophilic. In total, over 58% of the identified peptides were released from KRTs, and were from the four main regions of KRTs. The peptide features and the peptide release profiles from KRTs in SAH and GH were similar, which may provide a method for the identification of sustainable alternatives to replace the threatened SAH, and provide further evidence of the feasibility of using GH as a replacement for SAH based on their peptidomic analysis.

Identification of short-chain poly-3-hydroxybutyrates in Saiga horn extracts using LC-MS/MS.

Saiga horn extracts were analyzed with the goal of obtaining new information about compounds present in it. The purpose of this study is to find synthetic alternatives to Saiga horn extract, which is used in traditional Chinese medicine, by identifying potentially biologically active compounds in the extracts. Using high-performance liquid chromatography coupled with high-resolution mass spectrometry, we have been able to identify a series of short-chain polyhydroxybutyrates in alcoholic extracts of Saiga horn. Optimized high-performance liquid chromatography coupled with tandem mass spectrometry methods for analysis of short-chain poly-3-hydroxybutyrates were developed and subsequently applied to investigate Saiga horn extract for the presence of these compounds, which might explain its biological actions, particularly for its antipyretic and procoagulant properties.

Identification of rhinoceros keratin using direct analysis in real time time-of-flight mass spectrometry and multivariate statistical analysis.

RATIONALE: Trade in rhinoceros horn is regulated or banned internationally in recognition of its impact on wild populations worldwide. Enforcement of the laws and regulations depends on successfully identifying when violations occur, which is complicated by the presence of alternative/imitation rhinoceros horn keratin (e.g., bovid horn keratin). In this study, we assess the potential for Direct Analysis in Real Time (DART) ionization paired with Time-Of-Flight Mass Spectrometry (DART-TOFMS) to classify different keratin types from four taxonomic groups: rhinoceros, bovid, domestic horse, and pangolin. METHODS: The spectra of 156 keratin samples from all five rhinoceros species (horn keratin), eight genera of bovids (horn keratin), domestic horses (hoof keratin), and all extant species of pangolins (scale keratin) were collected. Fisher ratio analysis identified the most important ions that characterized each class and these ions were used for the training model, which consisted of 143 spectra. Kernel Discriminant Analysis (KDA) was used to classify the different groups. RESULTS: The spectra collected for each taxonomic group are distinctive. The chemotypes demonstrate that the spectra of rhinoceros, bovids, and domestic horse are similar to each other, whereas the chemotypes of pangolins show a different chemical profile. The model built by KDA resolved each taxonomic group: 95% of samples were correctly assigned using leave-one-out cross validation. The 13 blind samples not used in model development were all correctly classified to taxonomic source. CONCLUSIONS: DART-TOFMS appears to be a reliable approach for taxonomic identification of keratin. This analysis can be carried out with a small sliver of keratin, with minimal sample preparation, inexpensively and quickly, making it a potential valuable tool for identification of rhinoceros horn and other keratin types.

[Comparison on proteins of Saigae Tataricae Cornu and Caprae Hircus Cornu].

SDS-PAGE and LC-MS/MS were used to identify proteins in Saigae Tataricae Cornu (SAH) and Caprae Hircus Cornu (GH). Trypsin digestion peptides from SAH and GH were obtained by in-gel digestion, after which nano LC-LTQ/Orbitrap MS was used to identify the proteins in SAH and GH. As a result, in total 101 proteins and 140 proteins were identified form SAH and GH, respectively. There were 43 keratins (KRTs) and keratin-associated proteins (KAPs) identified, which account for 42.6% of the 101 proteins in SAH. The proportion of KRTs and KAPs in GH was 37.1%. The comparison between SAH and GH showed that the main common components in SAH and GH were structural molecule activity proteins, such as KRTs and KAPs. In the present study, we provide determination method and experimental data for investigating the material basis of SAH and GH, guiding the investigation on effective material basis and quality standard of animal horn derived TCMs.

Further evidence for sustainable alternatives to replace threatened animal horn based on quantitative proteomic analysis.

In the present study, a quantitative proteomic approach was used to analyze and compare the proteome in horns from endangered species (rhinoceros, Saiga antelope, and Tibetan antelope) and common species (yak, water buffalo, and goat) based on the isobaric tag for relative and absolute quantification techniques. In total, 591 proteins were identified, and 321 were quantified and categorized based on molecular function, cellular component, and biological process. Principal component analysis and hierarchical clustering analysis results based on differences in the amount of protein identified three major clusters, and proteins including transglutaminase, desmocollin, and elongation factors were selected as trait components from proteomic patterns of horn samples from different species. Quantitative proteomic analysis based strategies can therefore provide further evidence for sustainable alternatives to replace animal horn from threatened species.

Microstructure and mechanical properties of different keratinous horns.

Animal horns play an important role during intraspecific combat. This work investigates the microstructure and mechanical properties of horns from four representative ruminant species: the bighorn sheep (Ovis canadensis), domestic sheep (Ovis aries), mountain goat (Oreamnos americanus) and pronghorn (Antilocapra americana), aiming to understand the relation between evolved microstructures and mechanical properties. Microstructural similarity is found where disc-shaped keratin cells attach edge-to-edge along the growth direction of the horn core (longitudinal direction) forming a lamella; multiple lamellae are layered face to face along the impact direction (radial direction, perpendicular to horn core growth direction), forming a wavy pattern surrounding a common feature, the tubules. Differences among species include the number and shape of the tubules, the orientation of aligned lamellae and the shape of keratin cells. Water absorption tests reveal that the pronghorn horn has the largest water-absorbing ability due to the presence of nanopores in the keratin cells. The loading direction (compressive and tensile) and level of hydration vary among the horns from different species. The differences in mechanical properties among species may relate to their different fighting behaviours: high stiffness and strength in mountain goat to support the forces during stabbing; high tensile strength in pronghorn for interlocked pulling; impact energy absorption properties in domestic and bighorn sheep to protect the skull during butting. These design rules based on evolutionary modifications among species can be applied in synthetic materials to meet different mechanical requirements.

[Analysis of chondroitin sulfate content of Cervi Cornu Pantotrichum with different processing methods and different parts].

The differences and the variations of chondroitin sulfate content in different parts of Cervi Cornu Pantotrichum(CCP) with different processing methods were investigated. The chondroitin sulfate from velvet was extracted by dilute alkali-concentrated salt method. Next， the chondroitin sulfate was digested by chondroitinase ABC.The contents of total chondroitin sulfate and chondroitin sulfate A， B and C in the samples were determined by high performance liquid chromatography(HPLC).The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with freeze-drying processing is 14.13，11.99，1.74，0.32 g·kg⁻¹ï¼Œ respectively. The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with boiling processing is 10.71，8.97，2.21，1.40 g·kg⁻¹ï¼Œ respectively. The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP without blood is 12.47，9.47，2.64，0.07 g·kg⁻¹ï¼Œ respectively. And the content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with blood is 8.22，4.39，0.87，0.28 g·kg⁻¹ respectively. The results indicated that the chondroitin sulfate content in different processing methods was significantly different.The content of chondroitin sulfate in CCP with freeze-drying is higher than that in CCP with boiling processing.The content of chondroitin sulfate in CCP without blood is higher than that in CCP with blood. The chondroitin sulfate content in differerent paris of the velvet with the same processing methods was arranged from high to low as： wax slices， powder， gauze slices， bone slices.

Hierarchical structure and compressive deformation mechanisms of bighorn sheep (Ovis canadensis) horn.

Bighorn sheep (Ovis canadensis) rams hurl themselves at each other at speeds of ∼9 m/s (20 mph) to fight for dominance and mating rights. This necessitates impact resistance and energy absorption mechanisms, which stem from material-structure components in horns. In this study, the material hierarchical structure as well as correlations between the structure and mechanical properties are investigated. The major microstructural elements of horns are found as tubules and cell lamellae, which are oriented with (∼30°) angle with respect to each other. The cell lamellae contain keratin cells, in the shape of pancakes, possessing an average thickness of ∼2 µm and diameter of ∼20-30 µm. The morphology of keratin cells reveals the presence of keratin fibers and intermediate filaments with diameter of ∼200 nm and ∼12 nm, respectively, parallel to the cell surface. Quasi-static and high strain rate impact experiments, in different loading directions and hydration states, revealed a strong strain rate dependency for both dried and hydrated conditions. A strong anisotropy behavior was observed under impact for the dried state. The results show that the radial direction is the most preferable impact orientation because of its superior energy absorption. Detailed failure mechanisms under the aforementioned conditions are examined by bar impact recovery experiments. Shear banding, buckling of cell lamellae, and delamination in longitudinal and transverse direction were identified as the cause for strain softening under high strain rate impact. While collapse of tubules occurs in both quasi-static and impact tests, in radial and transverse directions, the former leads to more energy absorption and impact resistance. STATEMENT OF SIGNIFICANCE: Bighorn sheep (Ovis canadensis) horns show remarkable impact resistance and energy absorption when undergoing high speed impact during the intraspecific fights. The present work illustrates the hierarchical structure of bighorn sheep horn at different length scales and investigates the energy dissipation mechanisms under different strain rates, loading orientations and hydration states. These results demonstrate how horn dissipates large amounts of energy, thus provide a new path to fabricate energy absorbent and crashworthiness engineering materials.

Identification of four cornua by ultra-performance liquid chromatography with time-of-flight mass spectrometry coupled with principal component analysis.

An ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method coupled with principal component analysis was developed and applied to the identification of Cornu Antelopis, Cornu Bubali, Cornu Naemorhedi, and Cornu Bovis. The data obtained from the trypsin-digested samples were subjected to principal component analysis to classify these four cornua. Additionally, marker peptides of the cornua were determined by orthogonal partial least-squares discriminant analysis, and fragmentation tandem mass spectra of these marker peptides were evaluated. The results from this study indicate that the proposed method is reliable, and it has been successfully applied to the identification of variants of cornua commonly used in traditional Chinese medicine.

Characterization of Collagen Peptides in Elaphuri Davidiani Cornu Aqueous Extract with Proliferative Activity on Osteoblasts Using Nano-Liquid Chromatography in Tandem with Orbitrap Mass Spectrometry.

First documented in Shennong Bencao Jing (about 200 B.C.-200 A.D.), Elaphuri Davidiani Cornu (EDC) has been recorded for its effects in strengthening bones and balancing other aspects of overall health for approximately 2000 years. In the present study, our aim was to investigate which are the components of the active EDC fraction by a peptidomic strategy. We explored the extent to which EDC increases the proliferation of osteoblasts by measuring the elevations in osteonectin and type I collagen mRNA levels and characterized it using nano-flow liquid chromatography in tandem with orbitrap mass spectrometry. In total, 272 peptide sequences from collagens were determined. "Hot regions" in parent proteins determined by peptide heat maps which indicated that amino acid sequences in the regions might undergo proteolysis easily and generate peptides. Among the identified peptides, 90.2% were hydrophilic, and the molecular weight of 97.1% of identified peptides was lower than 2000 Da. According to these results, EDC collagen-derived peptides were easily analyzed and identified. Moreover, this methodology is feasible to characterize the active peptides matrices originated from collagen hydrolysates or some other animal horn- derived TCMs.

[Identification of water buffalo horn and its adulterants using COI barcode].

Bubali cornu (water buffalo horn) has been used as the substitute for Cornu rhinoceri asiatici (rhino horn) in clinical applications, and is the essential ingredient of Angong Niuhuang Wan. In recent years, there are a number of adulterants on the commercial herbal medicine markets. An efficient tool is required for species identification. In this study, 155 Bubali cornu samples have been taken from original animals and collected from commercial herbal medicine markets. 153 COI sequences have been successfully obtained from 155 samples through DNA extraction, PCR amplification, bidirectional sequencing and assembly. 93 COI sequences have been added to the DNA barcoding database of traditional Chinese animal medicine after validation using DNA barcoding GAP and tree-based methods. The species identification of the 62 commercial Bubali cornu medicines has been accomplished on the DNA barcoding system for identifying herbal medicine using the updated animal medicine database (www.tcmbarcode.cn). Except two samples failed to obtain COI sequences, 54.8% of the commercial Bubali cornu medicines were water buffalo horns and 29% were yak horns. Our results showed that yak horn was the major adulterant of Bubali cornu and the DNA barcoding method may accurately discriminate Bubali cornu and their adulterants. Therefore, we recommend that supervision on the herbal medicine markets should be strengthened with this new method to warren the effectiveness of herbal medicines.