WO2016153365A1

WO2016153365A1 - Naturally-derived compositions from calamus ornatus for anti-inflammatory applications

Info

Publication number: WO2016153365A1
Application number: PCT/PH2016/050002
Authority: WO
Inventors: Gracia Fe YU; Lourdes Cruz
Original assignee: Yu Gracia Fe
Priority date: 2015-03-24
Filing date: 2016-03-23
Publication date: 2016-09-29

Abstract

This invention pertains to derivatives of Calamus ornatus extracts and pure compound derivatives consistent with spirostanol saponins as selective inhibitors of cyclooxygenase 2 (COX-2). Activation of COX-2 is implicated with intense inflammation and pain, as well as other biological processes such as oncogenesis, angiogenesis and even for bowel movement. Hence, such activity of the compositions against COX-2 makes it a promising anti-inflammatory agent. With its different structure from the traditional COX-2 inhibitors, as well as the use of the source plant as a traditional food source, it is also likely that the invention might be safer to use, acutely or chronically.

Description

Naturally-derived compositions from Calamus ornatus for anti-inflammatory applications

Technical Field

This invention relates to the field of natural products, functional food and herbal- derived medicine, in particular to analgesics or pain-relieving substances.

Background of the Invention

Inflammatory conditions are very common medical problems. They include various forms of arthritides, sprains and strains, dysmenorrhea, toothaches, fractures and wounds, fatigue, post-surgical and dental operations, and local wound infections. Presently available drugs have been developed for inflammatory and pain-relieving conditions, but most of them are limited by possible serious side effects. The most commonly used are non-steroidal anti-inflammatory agents (NSAIDS), such as mefenamic acid, naproxen, ibuprofen, indomethacin, aspirin and diclofenac. However, these cyclooxygenase (COX)-nonspecific NSAIDs are strongly linked to ulcers and inflammation of the upper gastrointestinal tract (Curiel and Katz, 2013). Moreover, in susceptible individuals, they are associated with renal injury and failure. There are also non-NSAIDs anti-inflammatory agents; but there are also limitations on their use. Steroids, on the other hand, have several systemic complications, the most dangerous of which is the suppression of the immune system (Didonato et al., 1996). Paracetamol, which is a popularly used analgesic, is not as potent as the NSAIDs as a pain reliever (Brune and Neubert, 2001).

Presently, the major breakthrough in the development of anti-inflammatory drugs and analgesics is the emergence of the so-called COX-2-selective inhibitor drugs. These drugs include celecoxib (Celebrex), rofecoxib (Vioxx), etoricoxib (Arcoxia) and lumiracoxib (Prexige). The drugs block the inflammation-inducing COX-2 pathway with minimal inhibition of the COX-1 pathway, which, if inhibited, can result in gastric irritation and ulcers. Thus, COX-2- selective inhibitor drugs retain a potent anti-inflammatory activity with lesser side effects (Pellicano, 2014). However, there are several downsides for these drugs. First, they are very expensive. The traditional NSAIDs (such as mefenamic acid and naproxen) cost significantly more than the traditional NSAIDs (such as mefenamic acid and naproxen). Thus, for chronically ill patients and even occasional takers such effect on their budget could be draining. Second, they have potentially serious side effects. Celecoxib and rofecoxib are associated with adverse cardiac-related side effects, such as myocardial infarction (Brueggemann et al., 2009). This could be due to the secondary targeting of coxibs on calcium signalling of vascular smooth muscle cells that is separate from their cyclooxygenase inhibition. In contrast, the spirostanol saponins, which is radically different from coxibs, have been demonstrated to have cardioprotective effect in rats (Zhang et al. 2010); albeit, no study has been known in humans. Lumiracoxib has been pulled out of the market because of possible link to adverse liver- associated injuries (Fok et al., 2013).

The objective of the present invention is to provide anti-inflammatory and anti-pain compositions with COX-2-selective inhibitory properties that are less expensive and have better safety potential. It has been determined that the enzyme COX-2 is selectively inhibited by rattan limuran (Calamus ornatus Blume var. philippinensis) shoot components, with minimal inhibition of COX-1. COX-2 is induced anywhere in the body in response to inflammation causing symptoms such as pain and swelling; thus, inhibition of COX-2 activity leads to the alleviation of these inflammatory manifestations. In contrast to the traditional NSAIDs, the lack of COX-1 inhibition prevents the occurrence gastrointestinal injury. COX-1, constitutively expressed mainly in the digestive system and the genitourinary tract, has a major role in the protection of the mucosal linings (Pellicano, 2014). This explains why COX-2 selective inhibitory components have potent anti-inflammatory properties with lesser side effects. We present an invention that offers therapeutic and preventive solutions for the very common problems of inflammation and its associated pain. It offers several breakthroughs. First, it is derived from C. ornatus, an indigenous and endemic plant in the Philippines. The plant materials are food consumed by a Philippine ethnic group - Aytas - for centuries, attesting to its safety and availability as food. Typically, the plants grow in hills and forests away from polluted communities; thus the raw materials are cleaner and safer to consume. Second, the use of C. ornatus components as an anti-inflammatory agent is new. Although, the Aytas had indicated the anti-diarrheal effect of C. ornatus, we further explored to look for other potential health benefits of the plant, which eventually led to this discovery. Third, not satisfied with the mere crude extracts, the bioactive compounds were isolated, characterized and elucidated. Knowing the potent anti-inflammatory activity of the plant or its components made us offer the choices of making products in several forms, like functional food and drinks, food supplements or drugs. Fourth, after knowing the anti-intestinal motility effect of C. ornatus, which can lead to constipation after prolonged intake of C. ornatus preparations, we decided to add aqueous extract of G. binucao, which lessens the anti-motility effect of C. ornatus preparations. In addition, the G. binucao extract also neutralizes the bitter taste of C. ornatus. G. binucao is also a traditional herbal-derived food for Aytas and has been tested in the laboratory to be very safe for oral intake.

It is the belief of the inventors that there is no literature on the use of C. ornatus or G. binucao derivatives for anti-inflammatory or analgesic indications. Although the use of rattan is specified in patents to treat joint pains (CN102813867; CN103800618A), rattan is a generic plant term for about 600 species that does not point specifically to C. ornatus. Moreover, the aforementioned invention is unlikely to be C. ornatus, simply because the plant is not endemic in China. As for spirostanol saponins, several treatment indications are presented in the literature, such as Candida infection (MXMX/a/2007/014485), cardiovascular diseases (CN101035548), vulvovaginitis (US20050112218), hypoxia (CN102863501), and hyperglycemia (JP2005089419). However, the anti-inflammatory and/or analgesic action of the compounds have not been cited.

Summary of the Invention

The present invention is a composition containing derivatives from extracts of C. ornatus shoot, either in the form of crude extracts or in a chemically purified form of the active chemical compounds with COX2- specific properties. The chemical structures of the active compounds were determined to be consistent with spirostanol saponin- 1, spirostanol saponin 2, spirostanol saponin-3, and diosgenin. More importantly, the safety of the compositions has been verified through standard animal protocols, and has been considered to be very safe. Since the plant itself is used traditionally as food and medicine to humans, it is very likely that this plant material is safe for consumption. Part of the findings used for this invention has been published by the study group that includes the author (Yu et al., 2008). However, no data has come for human application, and the present formulation using G. binucao is not included.

Summary of Figures and Drawings

Figure 1 refers to the anti-inflammatory activity profile of the different proportions of C. ornatus - G. binucao extract formulation. The anti-inflammatory activity of formulations containing varying proportions of C. ornatus and G. binucao were compared with positive control (indomethacin) and negative control (normal saline solution [NSS]) using paw edema assay. Even after 4 hours, all formulations demonstrated significant anti-inflammatory activity in reference to the negative control. All formulations demonstrated comparable antiinflammatory activity among themselves and with the positive control. All points are the average value of duplicate assays (n=15 mice per assay) performed on different dates.

Figure 2 refers to the anti-inflammatory profile of G. binucao at 300, 150 and 75 mg/kg. The anti-inflammatory activity of varying concentrations of G. binucao was compared with positive control (indomethacin) and negative control (normal saline solution [NSS]) using paw edema assay. Even after 4 hours, all concentrations demonstrated significant anti-inflammatory activity in reference to the negative control. All concentrations demonstrated comparable anti-inflammatory activity among themselves and with the positive control. All points are the average value of duplicate assays (n=15 mice per assay) performed on different dates.

Figure 4 refers to the COX inhibitory activities of spirostanol saponin -1, -2, -3 and diosgenin at 28.2, 24.2, 21.2 and 60.4 μΜ, and the methanol extract at 250 ug/mL. Commercial antiinflammatory drugs, aspirin, celecoxib (Celebrex™) and rofecoxib (Vioxx™) were used as positive control and tested at 60 μΜ, 26 nM, and 32 nM concentrations, respectively. All compounds and the crude methanol extract from C. ornatus exhibited significant anti-inflammatory activity, which is comparable with celecoxib (CelebrexTM). The concentrations of positive controls used were based on their IC50. DMSO was used as solvent control. Percent inhibition was calculated with respect to DMSO control. Vertical bars represent average (n = 2) of two experiments +SD.

Description of the Invention

Processing of C. ornatus shoot as food or supplement

The collected edible shoots were immediately washed with water and boiled for 5 min. After cooling, the boiled shoots with water extracts were osterized and lyophilized. The lyophilized shoot was stored in the freezer. This boiled C. ornatus shoot can be eaten by itself or in combination with other food.

Processing of C. ornatus shoot with G. binucao extract as supplement

The G. binucao leaves were washed with water and boiled for 5 min. The water extract lyophilized was stored in the freezer. In order to neutralize the bitter taste of C. ornatus shoot, the G. binucao extract was incorporated producing a tea or smoothie.

As a tea, smoothie or health drink, the most preferred preparation is 3parts of C. ornatus and lpart of G. binucao to be combined. However, the proportions of C. ornatus: G. binucao may range from 1: 1 to 7: 1. Water and/or sugar or any sweetener can be added to suit the sweet taste of the consumer.

Collection and processing of C. ornatus shoot for methanol extracts and purification of bioactive substances

Shoots from rattan C. ornatus species are obtained. Edible shoot collected were lyophilized at once to avoid oxidation.

Preparation of Methanolic Extract of C. ornatus shoot

One hundred grams (lOOg) of lyophilized shoot of C. ornatus was extracted 3 consecutive times at least 6 hours each with methanol solvent. The extraction may also be done 1-4 times. The solvent-free methanol extract was obtained by rotary evaporation after which the extracts were stored in the freezer.

Purification of Bioactive Substances from C. ornatus extract

The 3g of methanol extract was passed through the gel column chromatography using 6:4 chloroform: methanol: solvent system to fractionate the different components. Depending on the capacity or targeted yield, the initial amount of methanol extract may vary. After rotary evaporation, each fraction was subjected to inflammatory testing. Fractions showing the bioactivity were pooled and were further isolated using the preparative thin layer chromatography preferably using 4:2 chloroform:methanol solvent system, but may range from 2: 1 to 1: 1. The TLC bands scraped were collected and re-extracted from the paper with the same solvent system. The solvents were removed using rotary evaporator and dissolved with chloroform:methanol combination for structural elucidation using NMR. Four pure compounds with COX-2 selective anti-inflammatory activity, diosgenin, spirostanol saponin- 1, -2 and -3 (from hereon also specified as pure compounds 1-4; 1- spirostanol saponin-1, 2- spirostanol saponin-2, 3- spirostanol saponin-3, 4-diosgenin), were afforded. The Markush structure involved a ubiquitous β-sitosterol glycoside covering all compounds and the possible side chain structures, more preferably wherein R is an H, a sugar moiety or a moiety of polymeric sugar (Figure 1). Thus, it is speculated that COX-2 inhibition is also possible with other substitutes in the R group, preferably with varying sugar moieties.

Evaluation of safety and dose range determination

It was determined that C. ornatus shoot has LD50 of 2,460 mg/kg body weight which denotes that this food is not harmful. The LD50 of boiled C. ornatus shoot: G. binucao extract formulation was more than 2000 mg/ kg body weight (OECD protocol) confirming the former LD50 for C. ornatus. Using the conversion factor of 0.0026 for humans from mouse data (Paget and Barnes, 1964), with 10-50% of the estimated LD50 in mice as basis, the estimated dose in human will be around 0.5-0.64 to 3.2 mg/kg.

Anti-inflammatory evaluation

To get the dose range of potency, the inflammatory effect of the different combinations of smoothie and G. binucao was determined (Figure 2). All combinations, Smoothie A (1: 1), Smoothie B (2: 1), Smoothie C (3: 1), Smoothie D (4: 1) and Smoothie E (5: 1) were found comparable to the positive control, indomethacin (2.5mg/kg body wt). The percent reduction of the inflammation was measured every after 30 min for 4 hours. Thirty minutes after the administration of the drug (indomethacin) to the mouse models the inflammation was reduced by 45.18% whereas the formulations reduced the inflammation at the range of 30.11% - 88.47%. After 2 hours, it was found that the formulations reduced the inflammation by 73.18 to 85.88% as compared to indomethacin (78.06%) which indicates that there was no significant difference in the activity between indomethacin and the formulations. A second administration of the drug and formulations was given after 2 hours. The measurement of paw thickness was continued. After 2.5 hours when the inflammation in untreated mice was at its peak, the inflammation was almost over because the indomethacin, Smoothie- 100, Smoothie A, Smoothie C, Smoothie E exhibited reduction by 91.92%, 89.08%, 85.66%, 84.16%, and 84.52%, respectively and 97.89%, 95.80%, 93.38%, 93.70% and 86.76% after 4 hours. Thus, it can be deduced that the formulations even at different proportions exhibited the similar effect to indomethacin, an anti-inflammatory drug.

G. binucao was likewise subjected to anti-inflammatory assay using Paw Method in mice (Figure 3). It can be deduced from Fig. 6 the significant effect of G. binucao even at the lowest dose of 75mg/kg of G. binucao extracts at different concentrations. At the dose range of 75 to 300 mg/mL, its activity profile was comparable to the positive control, indomethacin (49.37%) reducing the inflammation by an a average value of 51.26%. The percent increase in paw thickness of the negative control was measured 120%.

For the methanolic extract and the isolated compounds, the inhibitory activities of the extract and compounds were carried out against COX-1 and -2 enzymes according to the previously published procedures (Jayaprakasam et al., 2006) (Figure 4). The rate of oxygen consumption during the initial phase of the enzyme-mediated reaction, with arachidonic acid as substrate, was measured using a Model 5300 biological oxygen monitor (Yellow Spring Instrument Inc., Yellow Spring, OH). The test compounds, extract and positive controls were dissolved in DMSO separately. An aliquot of 10 uL of DMSO or test compounds or standards in DMSO was added to the reaction chamber containing 0.6 ml of 0.1 M Tris buffer (pH 7), 1 mM phenol and hemoglobin (17 ug). COX-1 or -2 enzymes (10 uL) were added to the chamber and incubated for 3 min. The reaction was initiated by the addition of arachidonic acid (10 uL of 1 mg/ml solution). Instantaneous inhibition was measured by using Quick Log Data acquisition and control computer software (Strawberry Tree Inc., Sunnyvale, CA, USA). The percent inhibition was calculated with respect to DMSO control. Pure compounds 1-4 were tested at 28.2, 24.2, 21.2 and 60.4 uM, respectively. Each sample was assayed twice and the standard deviation was calculated for n = 2. aspirin (60 1M), celecoxib (Celebrex™) (26 nM) and rofecoxib (Vioxx™) (32 nM) were used as positive controls.

Alternative embodiments

The preferred formulation of the invention is a combination of processed limuran C. ornatus shoot and aqueous leaf extract of G. binucao that makes the product acceptable, safe and more effective as a supplement and/or a drug. The presence of G. binucao extracts lessens the possibility of constipation due to C. ornatus and enhances the taste of the preparation. The C. ornatus: G. binucao proportion ranges from 1: 1 to 7:1 parts, most preferably at 3: 1. Alternatively, it is obvious that the bioactive derivatives are also obtainable from other parts of the plants, both from C. ornatus or from G. binucao.

Nevertheless, the compositions containing C. ornatus shoots or the derived bioactive compounds - spirostanol saponin -1, spirostanol saponin -2, spirostanol saponin -3, and diogenin - also produces similar anti-inflammatory effect regardless of the presence of G. binucao. However, G. binucao by itself has a significant anti- inflammatory effect, and may therefore be used in compositions that ameliorate inflammatory symptoms.

The derived products from the extracts and compounds from the invention can be in the form of capsules, tablets, syrup, liquid preparations, tea and functional food. Because of the high likelihood of safety of the extracts and compounds, the translation to the market can be immediate if provided as tea, herbal supplement or functional food. Processes related to drug-development are promising with potential significant contribution local economy, with implication for worldwide distribution.

In addition, the plants extracts and compounds can be added as fortificants to various food products, such as in bread or soy sauce) as functional food anti-inflammatory supplements. Citation List

Non-Patent Literature

Brueggemann LI, Mackie AR, Mani BK, Cribbs LL, Byron KL. Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles. Mol Pharmacol. 2009 Nov;76(5):1053-61.

Brune K, Neubert A. Pharmacokinetic and pharmacodynamic aspects of the ideal COX-2 inhibitor: a pharmacologist's perspective. Clin Exp Rheumatol. 2001 Nov-Dec;19(6 Suppl 25):S51-7.

Curiel RV, Katz JD. Mitigating the cardiovascular and renal effects of NSAIDs. Pain Med_¬ 2013 Dec;14 Suppl l:S23-8.

Didonato JA, Saatcioglu F, Karin M. Molecular mechanisms of immunosuppression and anti-inflammatory activities by glucocorticoids. Am J Respir Crit Care Med. 1996 Aug;154(2 Pt 2):S l l-5.

Fok KC, Bell CJ, Read RB, Eckstein RP, Jones BE. Lumiracoxib -induced cholestatic liver injury. Intern Med J. 2013 Jun;43(6):731-2.

Paget GE, Barnes J M. Toxicity tests in evaluation of drug activities pharmacometries (Laurence, D. R. and Bacharach, A. L. eds) Academic Press, London and New York, 1964.

Pellicano R. Gastrointestinal damage by non-steroidal anti-inflammatory drugs: updated clinical considerations. Minerva Gastroenterol Dietol. 2014 Dec;60(4):255-61.

Yu GF, Mulabagal V, Diyabalanage T, Hurtada WA, DeWitt DL, Nair MG. Non- nutritive functional agents in rattan- shoots, a food consumed by native people in the Philippines. Food Chemistry 15 Oct 2008 110(4):991-996.

Zhang S, , Yang R, Li H, , Yin Z, Zhou H, Li X, , Jin Y, Yang S. Separation and bio- activities of spirostanol saponin from Tribulus terrestris. Chem. Res. Chinese Universities 2010, 26(6): 915-921.

Patent Literature

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CN103800618 (A). Wang Y, et al. Oral pills for treating waist and leg arthralgia and preparation method thereof. 21.05.2014.

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Claims

A composition against inflammation and pain comprising derivatives from Calamus ornatus.

The composition according to claim 1 wherein the derivatives are obtained from the shoot of C. ornatus.

The composition according to claim 1 wherein the derivatives are obtained from non-shoot part of C. ornatus.

The composition according to claim 1 or 2 wherein the derivatives are methanolic extracts.

The composition according to claim 1 wherein the derivatives are purified compounds with a structure specified below;

wherein R is an H, a sugar moiety or is a moiety of polymeric sugar

The composition according to claim 5 wherein the derivative is spirostanol saponin- 1 with a R specified below:

The composition according to claim 5 wherein the derivative is spirostanol saponin-2 with a R specified below:

8. The composition according to claim 5 wherein the derivative is spirostanol saponin-3 with a R s ecified below;

9. The composition according to claim 5 wherein the derivative is spirostanol diosgenin with a R specified as H.

10. The composition according to claim 5 wherein it comprises a combination of any of the spirostanol saponins according to claims 6-9;

11. A method of processing plant parts of C. ornatus comprising of cleaning the shoots and breaking them down to smaller pieces.

12. The method according to claim 11 wherein the plant part is the shoot.

13. The method according to claim 11 wherein the shoots are lyophilized after cleaning and before breaking them down to smaller pieces.

14. Compositions that are derivatives of the methods according to claims 11-13.

15. Compositions specified according to claim 14 wherein the product is in a form of dried plant part for preparation of decoctions or tea.

16. Compositions specified according to claim 14 wherein the product is in a form of processed plant part for preparation of drinks and smoothies.

17. A composition against inflammation and pain comprising derivatives from Garcinia binucao.

18. The composition according to claim 17 wherein the derivatives are obtained from the leaves of G. binucao.

19. The composition according to claim 1 wherein the derivatives are obtained from non-leaf part of G. binucao.

20. The composition according to claim 17-18 wherein the derivatives are aqueous extracts.

21. A composition against inflammation and pain comprising a combination of derivatives from both C. ornatus and G. binucao.

22. The composition according to claim 21 wherein the derivatives of C. ornatus are from its methanolic extracts.

23. The composition according to claim 21 wherein the derivatives of C. ornatus are from its purified spirostanol saponins.

24. The composition according to claim 23 wherein the spirostanol saponin/s is/are as specified according to claims 5-10.

25. The composition according to claim 21 wherein the G. binucao derivatives are from its aqueous leaf extracts.

26. Use of compositions according to claim 1.

27. Use of compositions according to claim 17.

28. Use of compositions according to claim 21.