Revival of Complementary and Alternative Herbal Medicines

Herbal medicines, complementary or alternative medicines is a wide term for the therapies that are not part of standard care but it has many theories regarding efficacy based on personal experiences, history and common knowledge. It has long been used since ancient times since the beginning of human civilization. Its use had caught much attention in the early 1800s, with the development in the science of chemistry, a new era in pharmacotherapeutics and the use of active chemical ingredients in plants which were known to produce favorable therapeutic effects, were explored, active compounds were extracted, purified and their structure was revealed. This advancement paved the way towards modern pharmaceutical therapy. The modern drugs are based on these herbal medicines, after extracting the active and pure chemical compounds. Pharmacokinetics and physicochemical properties of the active ingredients was explored. It lead to the better understanding of efficacy and safety profile of these drugs and first choice for treatment of various diseases. At the same time, the herbal medicines were considered as secondarily important. After approximately two centuries, the use of herbal medicines have seen a revival globally both in developing as well as developed countries. In the past few years, the practice of using herbal medicines as an alternative and complementary health medicine has gained more importance. Herbal medicines are common for treatment of various ailments including cancer, digestive disorders, pain related disorders, neuropathic ailments and cardiac arrhythmias etc. Even it has been used by pregnant females and mostly perceived as safe. Its use has gained more attraction due to its ‘natural’ approach and lesser side effects. Their use if often overlooked but physicians should pay attention to these medicines. There is lack of familiarity, standardization of the drug components, unproven therapeutic effects in various diseases, unexplored toxicology, pharmacokinetics, drug-drug interactions, and compatibility in patients with varying medical, genetic and demographic history. There are serious concerns regarding the safety, efficacy and quality of herbal products and nutraceuticals. Accidental contamination and deliberate adulteration are assumed to be the main cause of the side effects. Much of the herbal medical knowledge is scattered in different regions of the world and mostly available at family, community and local level and mostly in any native languages. There is need of coherent sources, knowledge, and exploration of these medicines across the world. The herbal medicine has varying diversity in different geological regions and they should be investigated. There should be a regional or national body to control and approve the herbal medicines. Proper documentations on these medicines and food supplements should also be done.

Analysis of Metabolites from Healthy and Huanglongbing Infected Citrus Varieties of Pakistan

Citrus holds a prominent position in fruit’s world industry. Specifically, in Pakistan, citrus grown over 206,569 hectares with the gross annual production of 2.36 MT. Pakistan produced more than 30 different types of fruits, in which citrus is a leading fruit and constitutes 30% of total fruit production among all other fruits. More than 95% citrus fruits produced from the province Punjab and distributed through different value chains in domestic regions as well as in international fruit market. Pakistan citrus industry is a growing industry and dealing with many pre-harvest and post-harvest problems, which includes, bacterial and viral diseases, poor management and pest attack. Apart from all mentioned production problems, recent report according to Gulf Times stated that “Pakistan fetched more than $220mn through exporting 370,000 tons of citrus fruits to 58 different countries from December 2017 to May 2018, which increased the exporting rate up-to 14% from earlier period of exporting in 2016. It was also reported that 30,000 tons of Pakistani Mandarin exported to Indonesia last year, which is the biggest fruit market after Russia”. If we look on declining rate of citrus in Pakistan fruit industry, the major cause is of bacterial and viral diseases which covers the citrus tree in earlier ages and our poor management have yet no secure methods to overcome with these diseases in growing areas of Pakistan. Haunglongbing or Citrus Greening have major importance among all plant bacterial diseases which is responsible of declining production rate of citrus in Pakistan, as well as world widely. The main Protobectia (ɑ subdivision class), Candidatus Liberibecter is responsible for HLB disease, which mainly includes three different types: Candidatus Liberibecter asiaticus (CLas). Candidatus Liberibecter americanus (CLam) and Candidatus Liberibecter africanus (CLaf) according to dispersing of HLB to different countries. It is a gram negative, fastidious nature uncultured bacteria which grows in the phloem, sieve tubes elements of plant cells and generally transmitted through Psyllid, Diaphorina citri, which infested the bacterium mainly through grafting of citrus trees. The HLB bacterium moves unevenly in the midrib of leaves and distributed asymmetrical chlorosis on leaf cells which is the main symptom of this disease and mock to the zinc deficiency symptoms. It also affected the roots, bark (stem) and fruit (peel and pulp) of citrus fruits. The citrus fruit indicated green at the lower end and yellow underneath the peduncle after affecting this disease, with aborted seeds. Moreover, fruit flavor rapidly converted to bitter taste after infected to CLas. HLB mostly affecting the trees in 5-6 years age and covering all over the tree rapidly within a year. Most of the times, there are more than one bacterial and viral diseases also covers the citrus tree, i.e. CTV and citrus canker disease, which also show the symptoms similar to different nutrient deficiencies. Fifteen different types of citrus cultivars (healthy and HLB-affected) were selected for this study from Citrus Research Institute (CRI), Sargodha, Punjab, Pakistan. Citrus samples included, leaves and pulp and stored in 4°C container and transported to LCWU, Biotechnology Lab. After the confirmation of CLas-infection through conventional PCR, the citrus sample were Freeze dried (Lyophilized) for transporting to the United States. The level of infection in HLB-affected leaves samples were again reconfirmed at Microbiology Cell Sciences Department, USA, from the lyophilized leaf tissue (≤ 200 mg) through quantitative RT-PCR utilizing SYBR® Green and primer set LJ900 which produced an amplicon derived from the repeated copies of the prophage present within the Candidatus Liberibacter asiaticus (CLas) genome. Cycle threshold (Ct) values were determined and confirmed the healthy and infected samples. The range of Ct values for healthy to HLB-affected citrus samples ranged from 36.3 to no Ct detectable. For infected leaves, most copy number values were in the millions, with the highest being 4.50x106 (Ct=16.50). There were 3 exceptions ranging in copy number from 1.39x 103 (Ct=28.2) to 1.87x 105 (Ct=21.1). The HLB infection status was designated either positive or negative based on these results. After reconfirmation of HLB-infection in citrus samples by quantitative PCR, the first main analysis was the determination of the levels of zinc (Zn), phosphorous (P), and potassium (K) by inductively-coupled plasma mass spectrometry (ICP-MS). Pulp tissues contained lower levels of all three minerals regardless of the infection status. No significant change (Tukey’s t-test) was found in K levels associated with the HLB-affected leaves (p=0.7843) or pulp (p=0.0997). Phosphorous decreased 9% (p=0.0437) in leaves and increased 29% (p=0.0120) in pulp in HLB-affected samples compared to healthy. Zn showed a 31% (p<0.0001) decrease in infected leaves but no change in the pulp tissue (p=0.6728). These results explained the partitioning of Zn and P between leaves and fruit was differently affected by the HLB-infection status of the tree. However, no relationship between HLB infection status and K was observed. The next major part of this study was to determine the profile of vast range of targeted and untargeted metabolites from Global mass spectrometer analysis (LC-MS). And results indicated that there was a total of 13,752 features detected from all measurements of metabolites in leaves and fruit pulp (negative and positive ions) in the healthy and infected samples combined. Sixty five percent of the features were found in the negative ion-mode and 35% were noticed in positive ion-mode. The PLS-DA separations (known and unknown features) are highly significant for both negative and positive ions when leaf and pulp samples are combined; however, this separation is largely due to differences between leaves and pulp tissues. For the knowns, this conclusion is supported by the complete lack of separation between healthy and infected samples when the pulp is analyzed alone (negative ions p=0.92 and positive ions p=0.99). For leaves, there is a 52% probability that the separation is observed for the known negative ions; however, for the known positive ions, the probability that the separation is due to random features is 88% (p=0.88), similarly to the pulp values. For the unknown positive features, the PLS-DA separations of the leaf and pulp samples is largely based on the differences between pulp and leaves. In the case of the unknown negative features, the significance of the separations is better than those obtained with the positive ions. For unknown negative features in leaves, the probability that the model of separation may be random is 25% (p=0.25), while for the pulp samples, the probability for random separation is a 10% (p=0.10). Although the known positive ions in leaves is not useful (p=0.91), there is an approximately 50% chance that some of the known negative ions may contribute to the separation of healthy and HLB-infected samples. These both type of analysis (ICP-MS and LC-MS) gives complete range of profiles of metabolites and major macro/micro nutrients to future researchers in term of resorption of reallocation of minerals and metabolites with respect of Haunglongbing. Moreover, it gave more ground to targeted approach of unknown metabolites for future study. This is the first study in history of Pakistan which gives the metabolomic approaches of citrus cultivars infected with HLB to reader.