Lycium ruthenicum Murray derived exosome-like nanovesicles inhibit Aß-induced apoptosis in PC12 cells via MAPK and PI3K/AKT signaling pathways.

Plant-derived exosome-like nanovesicles (ELNs) are nano-sized vesicles extracted from edible plants. Lycium ruthenicum Murray (LRM) has been gaining increasing attention due to its nutritional and medicinal value, but the ELNs in LRM has not been reported. In this study, LRM-ELNs were obtained, and the proteins, lipids, microRNAs (miRNAs) and active components in LRM tissues and LRM-ELNs was analyzed by LC-MS/MS, LC-MS, high-throughput sequencing techniques, and physical and chemical analysis. LRM-ELNs can be uptaken by PC12 cells through macropinocytosis and caveolin-mediated endocytosis primarily. Transcriptomic and western blot experiments indicate that LRM-ELNs can inhibit Aß-induced apoptosis in PC12 cells through the MAPK and PI3K/AKT signaling pathways, with miRNAs playing a crucial role. These results indicate that LRM-ELNs have the protection effect on PC12 cells and can be considered as dietary supplements for alleviating neurodegenerative diseases.

Lycium ruthenicum water extract preserves retinal ganglion cells in chronic ocular hypertension mouse models.

Lycium ruthenicum Murray (LR), known as "black goji berry" or "black wolfberry", is widely utilized in chinese herbal medicine. LR fruit showed its antioxidant and/or anti-inflammation activity in treating cardiac injury, experimental colitis, nonalcoholic fatty liver disease, fatigue, and aging. Glaucoma is the leading cause of irreversible blindness. Besides elevated intraocular pressure (IOP), oxidative stress and neuroinflammation were recognized to contribute to the pathogenesis of glaucoma. This study investigated the treatment effects of LR water extract (LRE) on retinal ganglion cells (RGCs) threatened by sustained IOP elevation in a laser-induced chronic ocular hypertension (COH) mouse model and the DBA/2J mouse strain. The antioxidation and anti-inflammation effects of LRE were further tested in the H2O2-challenged immortalized microglial (IMG) cell line in vitro. LRE oral feeding (2 g/kg) preserved the function of RGCs and promoted their survival in both models mimicking glaucoma. LRE decreased 8-hydroxyguanosine (oxidative stress marker) expression in the retina. LRE reduced the number of Iba-1+ microglia in the retina of COH mice, but not in the DBA/2J mice. At the mRNA level, LRE reversed the COH induced HO-1 and SOD-2 overexpressions in the retina of COH mice. Further in vitro study demonstrated that LRE pretreatment to IMG cells could significantly reduce H2O2 induced oxidative stress through upregulation of GPX-4, Prdx-5, HO-1, and SOD-2. Our work demonstrated that daily oral intake of LRE can be used as a preventative/treatment agent to protect RGCs under high IOP stress probably through reducing oxidative stress and inhibiting microglial activation in the retina.

Protective effect and mechanism of Lycium ruthenicum Murray anthocyanins against retinal damage induced by blue light exposure.

Lycium ruthenicum Murray (LR) is a medicine and edible plant in Northwest China, and L. ruthenicum Murray anthocyanins (LRA) are green antioxidants with various pharmacological activities, such as antioxidant and anti-inflammatory activities. However, the protective effect and mechanism of LRA against retinal damage induced by blue light exposure are poorly understood. This study explored the protective effects and potential mechanisms of LRA on retinal damage induced by blue light exposure in vitro and in vivo. The results showed that LRA could ameliorate oxidative stress injury by activating the antioxidant stress nuclear factor-related factor 2 pathway, promoting the expression of phase II detoxification enzymes (HO-1, NQO1) and endogenous antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and reducing reactive oxygen species and malondialdehyde levels. Additionally, LRA could inhibit inflammatory response by decreasing the expression of blue light exposure-induced nuclear factor-κB (NF-κB) pathway-related proteins (NF-κB and p-IκBα), as well as interleukin (IL)-6, tumor necrosis factor-α, IL-1ß pro-inflammatory factors and pro-inflammatory chemokine VEGF, and increasing the expression of anti-inflammatory factor IL-10. Furthermore, LRA could ameliorate oxidative stress-induced apoptosis by upregulating Bcl-2 and downregulating Bax and Caspase-3 protein expression. All these results indicate that LRA can be used as an antioxidant dietary supplement for the treatment or prevention of retinal diseases.

Research Progress on Active Ingredients and Product Development of Lycium ruthenicum Murray.

Lycium ruthenicum Murray possesses significant applications in both food and medicine, including antioxidative, anti-tumor, anti-fatigue, anti-inflammatory, and various other effects. Consequently, there has been a surge in research endeavors dedicated to exploring its potential benefits, necessitating the organization and synthesis of these findings. This article systematically reviews the extraction and content determination methods of active substances such as polysaccharides, anthocyanins, flavonoids, and polyphenols in LRM in the past five years, as well as some active ingredient composition determination methods, biological activities, and product development. This review is divided into three main parts: extraction and determination methods, their bioactivity, and product development. Building upon prior research, we also delve into the economic and medicinal value of Lycium ruthenicum Murray, thereby contributing significantly to its further exploration and development. It is anticipated that this comprehensive review will serve as a valuable resource for advancing research on Lycium ruthenicum Murray.

Anthocyanins from Lycium ruthenicum Murray attenuates high-fat diet-induced hypercholesterolemia in ApoE-/- mice are related to the modulation of gut microbiota and the ratio of conjugated to unconjugated bile acids in fecal bile acid profile.

Previous findings showed that anthocyanins from Lycium ruthenicum Murray (ACN) reduced HFD-induced hypercholesterolemia by regulating gut microbiota, but the mechanism has not been fully understood. The objective of this research was to know whether the cholesterol-lowering impact of ACN in HFD-induced ApoE-/- mice is related to the gut microbiota-bile acid (BA) metabolism. Twenty-four male ApoE-/- mice were divided into three groups: the Control group, the HFD group, and the HFD + ACN group. Here, we showed that ACN intervention reduced HFD-induced body weight serum concentrations of TC and LDL-C and ameliorated lipid accumulation in the liver and adipose tissues. Besides, ACN altered gut microbiota composition in HFD-fed ApoE-/- mice. Moreover, UHPLC-MS/MS analysis revealed that ACN intervention significantly increased the ratio of conjugated to unconjugated BAs in feces induced by HFD, attributed to the increase in conjugated BAs and decrease in unconjugated BAs. Finally, the correlation analysis indicated that the above changes in fecal BA profile were linked with an increase in Bifidobacterium, Allobaculum and a decrease in Ileibacterium, Helicobacter, Rikenellaceae_RC9_gut_group, Blautia, Odoribacter, and Colidextribacter. In summary, ACN could alleviate HFD-induced hypercholesterolemia in ApoE-/- mice, which was associated with the improvement of gut microbiota and modulation of fecal BA profile.

Protective Effects of Lycium ruthenicum Murray against Acute Alcoholic Liver Disease in Mice via the Nrf2/HO-1/NF-κB Signaling Pathway.

Acute alcoholic liver disease (ALD) resulting from short-term heavy alcohol consumption has become a global health concern. Moreover, anthocyanins have attracted much attention for their ability to prevent oxidation and inflammation. The present work evaluates the protective effects of Lycium ruthenicum Murray (LRM) against ALD and explores the possible underlying mechanism involved. The total anthocyanin content in LRM was 43.64 ± 9.28 Pt g/100 g dry weight. Mice were orally administered 50, 125, or 375 mg LRM/kg body weight (BW) for 21 days. On days 18-21, mice were orally administered 15 mL of ethanol/kg BW. Markers of liver damage, oxidative stress, and inflammation were examined. Furthermore, the modulatory effect of LRM on Nrf2/HO-1/NF-κB pathway molecules was evaluated through quantitative reverse transcription polymerase chain reaction (RT‒qPCR) and immunohistochemistry analyses. The difference between the groups indicated that LRM improved liver histopathology and the liver index, decreased aspartate transaminase, alanine transaminase, malondialdehyde, reactive oxygen species, IL-6, TNF-α, and IL-1ß expression, but elevated superoxide dismutase, catalase, and glutathione-s-transferase levels. Moreover, LRM upregulated Nrf2 and Ho-1 but downregulated Nf-κb and Tnf-α genes at the transcript level. In summary, LRM alleviated ethanol-induced ALD in mice by reducing oxidative damage and associated inflammatory responses. LRM protects against ALD by reducing damage factors and enhancing defense factors, especially via the Nrf2/HO-1/NF-κB pathway. Thus, LRM has application potential in ALD prophylaxis and treatment.

Anthocyanins from Lycium ruthenicum Murray Mitigate Cadmium-Induced Oxidative Stress and Testicular Toxicity by Activating the Keap1/Nrf2 Signaling Pathway.

Cadmium (Cd) is a hazardous heavy metal environmental pollutant that has carcinogenic, teratogenic, and mutagenic properties. Excessive exposure to Cd can induce oxidative stress, which greatly harms the male reproductive system. Anthocyanins have remarkable antioxidative, anti-inflammatory, and anti-stress properties. In this study, we investigated the effects of anthocyanins and the underlying mechanisms through which anthocyanins mitigate Cd-induced reproductive damage. We isolated and purified Lycium ruthenicum Murray anthocyanin extract (LAE) and performed UHPLC-MS/MS to identify 30 different anthocyanins. We established an ICR mouse Cd injury model by administering 5 mg/kg/day CdCl2 for 28 consecutive days. LAE at 500 mg/kg/day effectively ameliorated testicular damage and preserved spermatogenesis. The mice in the LAE-treated group had elevated testosterone and inhibin B levels. Additionally, the treatment restored the activity of antioxidant enzymes, including T-SOD, CAT, and GR, and substantially increased the levels of the non-enzymatic antioxidant GSH. Research findings indicate that LAE can activate the SIRT1/Nrf2/Keap1 antioxidant pathway. This activation is achieved through the upregulation of both the SIRT1 gene and protein levels, leading to the deacetylation of Nrf2. Moreover, LAE reduces the expression of Keap1, alleviating its inhibitory effect on Nrf2. This, in turn, facilitates the uncoupling process, promoting the translocation of Nrf2 to the nucleus, where it governs downstream expression, including that of HO-1 and GPX1. LAE effectively mitigated toxicity to the reproductive system associated with exposure to the heavy metal Cd by alleviating oxidative stress in the testes.

Black Goji Berry (Lycium ruthenicum Murray): A Review of Its Pharmacological Activity.

Lycium ruthenicum Murray (LRM; commonly known as black goji berry or black wolfberry), a plant in the Solanaceae family, grows in the deserts of China's Qinghai-Tibet plateau. LRM is widely consumed in traditional Chinese medicine, and its fruits are frequently used as herbal remedies to treat heart disease, fatigue, inflammation, and other conditions. Many studies have reported that LRM is rich in functional phytochemicals, such as anthocyanins and polysaccharides, and has various pharmacological actions. This article reviews research on the biological and pharmacological effects of the constituents of LRM fruits. LRM has various pharmacological properties, such as antioxidant, anti-inflammatory, anti-radiation, immune-enhancing, anti-tumor, and protective effects. LRM has much promise as a dietary supplement for preventing many types of chronic metabolic disease.

Inhibitory effects of the anthocyanins from Lycium ruthenicum Murray on angiotensin-I-converting enzyme: in vitro and molecular docking studies.

BACKGROUND: Lycium ruthenicum Murray (LRM), a perennial shrub plant belonging to the Solanaceae family, is rich in anthocyanins, which have anti-inflammatory, antioxidant, lipid-lowering, intestinal flora regulating, and other pharmacological qualities. This study was primarily aimed to investigate the inhibitory effect of different anthocyanin purities from LRM on angiotensin-I-converting enzyme (ACE) activity in vitro. Moreover, the inhibitory mechanism was further analyzed by molecular docking technology. RESULTS: Two main anthocyanin isomers were identified by ultra-performance liquid chromatography-tandem mass spectrometry and proton/carbon-13 nuclear magnetic resonance, namely petunidin-3-O-[rhamnopyranosyl-(trans-p-coumaroyl)]-5-O-(ß-d-glucopyranoside) (trans-Pt3R5G) and petunidin-3-O-[rhamnopyranosyl-(cis-p-coumaroyl)]-5-O-(ß-d-glucopyranoside) (cis-Pt3R5G), with a molar ratio of 9:1. Three purification grades of Pt3R5G all showed excellent inhibitory effects on ACE, with the half maximal inhibitory concentration (IC50 ) values being 0.562, 0.421, and 0.106 mg·mL-1 . Increasing the purity may reduce the IC50 within a certain concentration range. An enzymatic kinetic experiment showed that the inhibitory effect of Pt3R5G on ACE was reversible and non-competitive: Pt3R5G and substrate were not in competition for the active sites of ACE. Molecular docking technology further revealed the possible mechanism was that Pt3R5G and ACE amino acid residues were interacting by hydrogen bonds to exert the inhibitory effect. CONCLUSION: The results indicated that Pt3R5G from LRM was highly effective at inhibiting ACE activity in vitro, with the hydrogen bonds of Pt3R5G and ACE amino acid residues exerting the inhibition. As a potential plant-based ACE inhibitor, Pt3R5G can be used as a functional ingredient for antihypertensive effects. © 2023 Society of Chemical Industry.

Anthocyanins from Lycium ruthenicum Murray Ameliorated High-Fructose Diet-Induced Neuroinflammation through the Promotion of the Integrity of the Intestinal Barrier and the Proliferation of Lactobacillus.

In the present study, we found that anthocyanins from Lycium ruthenicum Murray (ACN) potently ameliorated a high-fructose diet (HFrD)-induced neuroinflammation in mice. ACN improved the integrity of the intestinal barrier and suppressed the toll-like receptor 4 (TLR4) signaling pathway to ameliorate the neuroinflammation, which was verified by Tlr4-/- mice. Furthermore, ACN could modulate the HFrD-induced dysbiosis of gut microbiota. The fecal microbiota transplantation from ACN-induced mice was sufficient to attenuate the neuroinflammation, while the amelioration of neuroinflammation by ACN was blocked upon gut microbiota depletion. In addition, ACN-induced increment of the relative abundance of Lactobacillus might be responsible for the alleviation of the neuroinflammation, which was further confirmed in the promoting effect of ACN on the growth of Lactobacillus in vitro. Overall, these results provided the evidence of a comprehensive cross-talk mechanism between ACN and neuroinflammation in HFrD-fed mice, which was mediated by reducing gut microbiota dysbiosis and maintaining the intestinal barrier integrity.

In Situ Stability of Anthocyanins in Lycium ruthenicum Murray.

In this research, the effects of drying method, storage temperature, and color protector glucose on anthocyanin preservation in the Lycium ruthenicum Murr. fruit were studied. Compared with hot-air drying, vacuum freeze-drying preserved about 5.8-fold more anthocyanins. The half-life of anthocyanins in the freeze-dried fruit samples with glucose was 3.6 days, 1.8 days, and 1.7 days at 4 °C, 20 °C, and 37 °C, respectively. On the other hand, the half-life values without glucose addition were 2.2 days, 2.3 days, and 2.1 days at each temperature, respectively, indicating that glucose protected anthocyanins at low temperature. The composition and contents of anthocyanins and anthocyanidins in the freeze-dried Lycium ruthenicum Murr., stored for 20 days, were investigated with a HPLC-MS/MS setup. It was found that most anthocyanidins in Lycium ruthenicum Murr. are linked with coumaroyl glucose to form anthocyanins, while glycosylated and acetyl-glycosylated anthocyanins were also detected. Five anthocyanidins were detected: delphinidin, cyanidin, petunidin, malvidin, and peonidin, and delphinidin accounts for about half of the total amount of anthocyanidins. It is much more economic to conserve anthocyanins in situ with freeze-drying methods and to store the fruits at low temperatures with glucose.

Anthocyanins in Lycium ruthenicum Murray reduce nicotine withdrawal-induced anxiety and craving in mice.

Lycium ruthenicum Murray is widely used in traditional Chinese medicine and is believed to have antimicrobial, antioxidant, and anti-fatigue effects. Anthocyanins are considered to be one of the main active components. The previous work by our research team found that the anthocyanins in Lycium ruthenicum extract (ALRM) produce a stable anti-anxiety effect. The mechanisms of action include reducing the level of corticotropin-releasing factor (CRF) as well as regulating extracellular signal-regulated kinase/mitogen activation, protein kinase (ERK/MAPK) pathways, and others, all of which are related to the mechanisms of nicotine addiction. To investigate the effects of ALRM on anxiety and craving behavior after nicotine withdrawal, the components of ALRM were analyzed using the UPLC-Orbitrap MS method. The effects of ALRM on anxiety behavior induced by nicotine withdrawal were investigated in mice using the elevated plus maze (EPM) and light-dark box (LDB) tests. The effects of ALRM on craving behavior after nicotine withdrawal were further investigated using the conditional place preference (CPP) test. The EPM and LDB tests demonstrated that ALRM could alleviate the anxiety behavior induced by nicotine withdrawal and reduce nicotine craving in mice. Based on the identified ALRM components, the network pharmacology method was used to predict the mechanism of ALRM alleviating anxiety after nicotine withdrawal in mice. It was speculated that ALRM was involved in the production and transmission of dopamine, choline, and other nervous system functions and exhibited a potential role in treating nicotine addiction.

The Main Anthocyanin Monomer from Lycium ruthenicum Murray Fruit Mediates Obesity via Modulating the Gut Microbiota and Improving the Intestinal Barrier.

Anthocyanins have been shown to exert certain antiobesity properties, but the specific relationship between anthocyanin-induced beneficial effects and the gut microbiota remains unclear. Petunidin-3-O-[rhamnopyranosyl-(trans-p-coumaroyl)]-5-O-(ß-D-glucopyranoside) (P3G) is the main anthocyanin monomer from the fruit of Lycium ruthenicum Murray. Therefore, in this study, we investigated the antiobesity and remodeling effects of P3G on gut microbiota through a high-fat diet (HFD)-induced obesity mouse model and a fecal microbiota transplantation experiment. P3G was found to reduce body weight gain, fat accumulation, and liver steatosis in HFD-induced obese mice. Moreover, supplementation with P3G alleviated the HFD-induced imbalance in gut microbiota composition, and transferring the P3G-regulated gut microbiota to recipient mice provided comparable protection against obesity. This is the first time evidence is provided that P3G has an antiobesity effect by changing the intestinal microbiota. Our present data highlight a link between P3G intervention and enhancement in gut barrier integrity. This may be a promising option for obesity prevention.

Petunidin-Based Anthocyanin Relieves Oxygen Stress in Lactobacillus plantarum ST-III.

Application of probiotics in the food industry has been hampered by their sensitivity to challenging conditions that reduce their vitality in food matrices. A lot of attempts have been made to promote the growth of these probiotics in the aspect of nutrition demands. Among the other adverse conditions, oxygen stress can restrict the growth of probiotics and has not yet been paid enough attention to. In this study, the effect of a petunidin-based anthocyanin (ACN) on the growth of probiotic Lactobacillus plantarum ST-III was investigated under oxygen stress. The growth of ST-III was analyzed through spot assay on agar plates as well as plating-based enumeration of the viable cells in the liquid culture. Results indicated that ACN could efficiently improve the growth of ST-III under oxygen stress, whereas no effect was observed in the absence of oxygen stress. Further investigations indicated that ACN reduced the oxido-reduction potential of the culture; meanwhile, it exerted a positive transcriptional regulation on the thioredoxin system of ST-III, leading to a decrease in reactive oxygen species accumulation within the cells. Moreover, ACN enabled the growth of ST-III in reconstituted skim milk and promoted the formation of milk clots. These results revealed the role of a petunidin-based ACN in oxygen stress relief and highlighted its potential in manufacture and preservation of L. plantarum-based dairy products.

Effects of long-term intake of anthocyanins from Lycium ruthenicum Murray on the organism health and gut microbiota in vivo.

The relationship between diet, especially polyphenols, and health has been receiving increasing attention. Polyphenols were degraded by gut microbiota into metabolites and acted on the body to exert many bioactivities from several targets such as antioxidative stress, anti-inflammation, intestinal barrier and gut microbiota modulation. After long-term treatment of mice with anthocyanins from Lycium ruthenicum Murray (ACN), antioxidant status in liver (T-AOC, T-SOD, CAT, GSH and GSH-Px were increased and AST, ALT, ALP and MDA were decreased), anti-inflammatory status in colon (the expression of mRNA of iNos, Cox-2, Tnf-α, Il-6, Il-1ß and Ifn-γ were significantly reduced), intestinal barrier (the expression of mRNA of Zo-1, Occludin, Claudin-1 and Muc1 were significantly increased) and gut microbiota (Barnesiella, Alistipes, Eisenbergiella, Coprobacter and Odoribacter were proliferated) were all regulated in ACN group. Meanwhile, the content of short-chain fatty acids in cecal contents and feces were increased. Taken together, long-term intake of ACN could promote organism healthy and these results have important implications for the development of ACN as a functional food ingredient.

Gut microbiota modulation and anti-inflammatory properties of anthocyanins from the fruits of Lycium ruthenicum Murray in dextran sodium sulfate-induced colitis in mice.

In the present study, the therapeutic effects of crude anthocyanins (ACN) from the fruits of Lycium ruthenicum Murray and the main monomer of ACN, petunidin 3-O-[rhamnopyranosyl-(trans-p-coumaroyl)]-5-O-[ß-d-glucopyranoside] (P3G), on the dextran sodium sulfate (DSS)-induced colitis in mice were investigated. Both ACN and P3G showed intestinal anti-inflammatory effects, evidenced by restoration of various physical signs (body weight, feed quantity, solid fecal weight and colon length were increased, and DAI score was decreased), reduction of the expression of proinflammatory cytokines and related mRNA (such as TNF-α, IL-6, IL-1ß and IFN-γ), and promotion of the intestinal barrier function by histological and immunofluorescence analysis (proteins such as ZO-1, occludin and claudin-1 were increased). Furthermore, the effects on gut microbiota community of DSS-induced colitis in mice have been investigated. It was found that Porphyromonadaceae, Helicobacter, Parasutterella, Parabacteroides, Oscillibacter and Lachnospiraceae were the key bacteria associated with inflammatory bowel disease. Taken together, P3G and ACN ameliorated DSS-induced colitis in mice through three aspects including blocking proinflammatory cytokines, increasing tight junction protein and modulating gut microbiota. What's more, P3G showed better anti-inflammatory effects than ACN.

Rapid qualitative and quantitative analyses of eighteen phenolic compounds from Lycium ruthenicum Murray by UPLC-Q-Orbitrap MS and their antioxidant activity.

Lycium ruthenicum Murray (LR) is a functional food, and it has long been used in traditional folk medicine. However, detailed qualitative and quantitative analyses related to its phenolic compounds remains scarce. This work reports, for the first time, the establishment of a rapid method for simultaneous identification and quantification of 25 phenolic compounds by UPLC-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS). This method was validated by LODs, LOQs, precision, repeatability, stability, mean recovery, recovery range and RSD. The confirmed method was applied to the analysis of phenolic compounds in LR. Finally, 18 phenolic compounds in LR were qualitatively and quantitatively analyzed. Among them, 11 constituents were detected for the first time, which included two flavonoids (catechin and naringenin) and seven phenolic acids (gallic acid, vanillic acid, 2,4-dihydroxybenzoic acid, veratronic acid, benzoic acid, ellagic acid and salicylic acid). Moreover, Phloretin and protocatechuate, belonging to the dihydrochalcone flavonoid and protocatechuic acid respectively, were also identified and quantified. The total phenolics content (20.17 ±â€¯2.82 mg/g) and the total anthocyanin content (147.43 ±â€¯1.81 mg/g) were determined. In addition, the antioxidant activities of the LR extract were evaluated through 2,2-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, ferric reducing antioxidant power (FRAP) and total antioxidant activity (T-AOC) assays.

Comparison and multivariate statistical analysis of anthocyanin composition in Lycium ruthenicum Murray from different regions to trace geographical origins: The case of China.

Anthocyanin composition in forty-five Lycium ruthenicum Murray (LRM) samples grown in China was identified by high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS) and quantified by HPLC with a diode array detector (HPLC-DAD). The results showed that the overall pattern of anthocyanin composition of LRM from different provinces was the same, while the individual and total anthocyanin concentrations, were significantly different, indicating an important impact of geographical origin on anthocyanin composition, which can be considered as credible indices for LRM classification. Principal component analysis (PCA) and linear discriminant analysis (LDA) were applied to develop discrimination models for the anthocyanin concentrations. PCA clearly separated the LRM based on its geographical origins. LDA satisfactorily categorized the samples by providing a 100% success rate based on geographical origins. The results obtained could be used to trace the geographical origin of LRM.