Cancer is emerging as one of the most serious life threatening diseases in the modern era and its growing prevalence means that it is of immediate worldwide concern.(1) Cancer is a disease characterised by an abnormal growth of cells in which the proliferation of normal cells divide uncontrollably. This can progress to neighbouring tissues and vital organs. (2) Synthetic drugs have made a tremendous contribution to various forms of cancer. However, because of their toxic side-effects and not being able to fulfil their objective of actually controlling tumour growth, the search for the ultimate cure for cancer has led researchers to investigate traditional Chinese herbal medicines (CHMs).(1)(3)
The successful development of anticancer agents from natural products such as vinca alkaloids e.g. vinblastine and vincristine derived from Cantharanthus roseus (2) and taxol, such as paclitaxel isolated from the Pacific Yew tree have allowed research to explore beyond the field of conventional medicine and return to the roots of traditional CHMs.
There has been some contribution to cancer chemotherapy from the East in the use of Chinese herbs. CHMs have been used for thousands of years and they are often used as an alternative treatment to orthodox medicines in the West. (4) CHMs have been used to prevent and treat many kinds of diseases in China, especially autoimmune diseases and tumours. (5),(6) Supporting evidence found that some CHMs have several immunodulatory activities that act on the main players of the immune system, such as B cells, T cells and macrophages and therefore offer promising evidence for the efficacy of medicinal herbs in these diseases.(5)
Oldenlandia diffusa (OD) is a member of the Rabiceae family. (1) It appears to have tremendous use all over the world including countries like Bangladesh and is one of the common herbs used in southern China for malignant tumours like colon cancer and other diseases such as appendicitis, hepatitis, tonsillitis and urethral infection as studied by Xu et al. (7) Evidence from Wong et al found that OD in vivo was able to significantly inhibit the growth of renal carcinoma cells in mice and enhance the effect of macrophage function.(6) However, there are few of these in vivo studies supporting its effects. Sadava et al provided evidence that OD has cytotoxic effects on many cell lines including both a lung cancer cell line and a multi-drug resistant (MDR) lung carcinoma cell line. The study also indicate that apoptosis is the pharmacological mode of action of OD. (6)
A study by Zhang and Song found the efficacy of a concoction of CHMs including OD in treatment of advance prostate cancer. However, because of the complex nature of the CHM that was used in the study cannot be 100% sure that OD exhibit any cytotoxic effect to prostate cancer cell. (8)
Extraction of OD using various solvents such as water, chloroform, ethanol and methanol gives different constituents and measures of anti-tumour activity. A study by Gupa et al found that the growths of 8 different cell lines were inhibited by the water extract of OD. (4) On the other hand, conflicting studies regarding the cytotoxic of the methanol extract of OD were found. (9) Liang et al found no anti-proliferative effect of the methanol extract of OD, whilst a study by Kim et al found that it exhibited a significant anti-tumour activity. (10) The differences may be due to the duration of OD in methanol - Liang et al refluxed OD in methanol for 4 hours, whereas Kim et al extracted theirs at room temperature for 2 weeks. The differences are multifactorial, and can explain the variation in anti-proliferative effect of the methanol (Liang's refluxing may have inactivated the cytotoxic constitutents, and Kim's longer extraction may have led to a higher concentration). Yadav et al found that the ethanol extract of OD (produced by overnight shaking at room temperature) evokes cancer cells apoptosis.(11) In practice OD is usually given as a tea so the water extract would better represent how people in China would ingest OD.(4)
Isolation of OD has found a high abundance of oleanolic acid (OA) and ursolic acid (UA) both of which are pentacyclic tritepenoids that are widely found in plants. (7) Andersson et al and Ovesna et al found that OA may be a MDR agent as studies have shown apoptosis in MDR Vincristine resistance cell line. (7) UA main component of methanol extract was not found in aqueous extract of OD showed significant anti-tumour activity Kim et al). Oguro et al found that OA was able to prevent 12-tetradecanyolphorbol-13 acetate TPA- induced carcinogenesis in animal studies supporting its anti-tumour activity. (7) Similarly Cha et al found that UA mode of action inhibits invasive tumours and metastasis through matrix metalloproteinase. (12) Other constituents of OD, such as iridoid glucosides, have also been shown to have a range of pharmacological activity including DNA polymerase inhibition and antineoplastic action. (9) Despite the limited data relating to iriod glycoside to the anti-tumour activity even though it is the main component of OD, stronger evidence suggest it may be useful in animals suffered from high cholesterol.
Chromatography involves the separation of compounds based on the interaction between the mobile phase and the stationary phase. (13) Between the periods of 1964-1965, the development of High Performance Liquid Chromatography (HPLC) became one of the branches of chromatography. (14) HPLC technique is a powerful analytical technique that has made a huge impact in analytical chemistry and allows rapid identification of complex compounds like CHMs. (15) It has been clearly recognised that there is a need for sensitive, selective detection and separating techniques such as HPLC. (16) HPLC has advantages over Gas Chromatography GC in terms of the speed of analysing compounds and efficiency of separation. (14) Gas Chromatography is also unsuitable because many organic compounds are too unstable or not volatile enough for this technique. (14)
The main limitation of HPLC is the inability to identify the compounds solely based on the retention time. Many compounds can have a similar retention time, so it is not a unique identifier. The combination of HPLC with mass spectroscopy allows compounds to be first separated and the retention time determines the identity of the compound based on its molecular weight. (16) Some researchers have validated HPLC methods for this purpose and found it to be a very useful technique (17).
Ganbold et al have successfully separated OD into 11 fractions, Fragment 9 (F9), in particular, having the most peaks and showing the greatest cytotoxic activity against human leukocyte-(HL)-60 and colon carcinoma (CaCo2) cells. They further separated F9 by using HPLC-LCMS to identify 2 of the 8 constituents as UA and OA. However, further separation is required to identify the remaining constituents of F9. (6)
Aim and Objectives
The aim of the study is to separate and identify fraction F9 of OD.
Objectives:
To carry out a water extraction of the herb OD and use HPLC to further analyse F9 by varying mobile phases, column types and HPLC machine and identify it using LC-MS
Determine the IC50 and LD50 of OD in human colon carcinoma (Caco2) and other cancer cell line
Materials and Method
Decoction preparation- (To carry out the water extraction of OD)
The herb OD will be given by Professor Bo-Ying Ma. 5g of OD were cut into 1-1.5mm pieces and boiled with 50ml deionised water, refluxing for 45minutes. After cooling, the decoction was centrifuged at 3000rpm for 20 minutes and filtered through 0.45um filter. The decoction was then made up to 50ml with deionised water.
HPLC and LC-MS analysis
HPLC:
Varian chromatography systems
Mobile phase system:
0-10 min 100% ammonium acetate buffer
10-40 min 100% → 50% ammonium acetate buffer
40-50 min 50% → 0% ammonium acetate buffer
50-51 min 0% → 100% ammonium acetate buffer
51-60 min 100% ammonium acetate buffer
Software:
Galaxie Chromatography Workstation version 1.9.3.2
Grade:
Sigma
Column:
Analytical column
Brand:
Phenomenex
P/No:
00G - 4448- EO
Desc:
Luna 5u PFP (2) 100A
Size:
250 x 4.60mm
Mobile phase:
Ammonium acetate buffer (pH 6.5) / methanol
Wavelength
226nm
Injection volume:
20ul (analytical column)
Flow rate:
1ml (analytical column)Structures and identity of OD is determined by LC-MS
Cell Culture- Once the effective constituents of OD have been obtained the LD50 and IC50 will be measured.
Time Frame / Likely Outcomes: To achieve the decoction preparation and the HPLC- LCMS analysis and to obtain UA, OA and any other constituents in F9 before the Christmas Holiday. Beginning of 2nd semester to start cell culture and determine IC50 and LC50 of OD. By March all results will have been analysed and discussion of the findings and putting the complete project together before the deadline.
