Hypertension represents a global health problem due to its increasing incidence worldwide, as well as the medical complications that can lead to patient death. The compound 3-n-butylphthalide (NBP) possesses various therapeutic properties, one of which is its potential antihypertensive effect, making it of great interest to the medical field. Traditional methods for obtaining this compound, such as chemical synthesis and extraction with organic solvents, exist. However, limitations related to their efficiency, yield, and environmental impact raise concerns about their sustainability, highlighting the need to develop an alternative production method. For this reason, the bioproduction of NBP is proposed, establishing an experiment aimed at achieving high yields at the laboratory level using genetically modified organisms (GMOs). Subsequently, the scale-up of NBP production in a bioreactor is planned. Ultimately, the goal is to explore the biotechnological potential of this production method for industrial and pharmaceutical applications.
Hypertension occurs when the pressure in blood vessels is too high, making it a chronic and serious medical condition. Moreover, it is a major risk factor for the development of cardiovascular diseases, which are the leading cause of death worldwide.
According to the World Health Organization (WHO), the global incidence of hypertension among adults (aged 30 to 79) has doubled—from 650 million to 1.3 billion—over a 30-year period (1990–2019). In Ecuador, according to the STEPS survey, 19.8% of the adult population (aged 18 to 65) has been diagnosed with hypertension. Furthermore, the Pan American Health Organization (PAHO) reports that one in five Ecuadorians suffers from this condition.
Currently, hypertension can be treated and managed with various types of medications such as diuretics, vasodilators, calcium channel blockers, renin inhibitors, and angiotensin-converting enzyme inhibitors. However, the daily use of these medications is associated with numerous side effects, including edema, muscle cramps, skin rashes, vomiting, and kidney failure. As a result, many patients discontinue their treatment and turn to natural medicine based on medicinal plants.
The compound 3-n-butylphthalide (NBP) is an active component found in plants of the Apiaceae family. This compound belongs to the phthalide group, which has neuroprotective properties and is used to treat ischemic stroke. Many studies have shown that NBP derived from celery can help lower blood pressure by acting as a diuretic and vasodilator. One possible mechanism of action involves the blocking of calcium entry into cells or the release of calcium from the sarcoplasmic reticulum.
Organic solvent extraction and chemical synthesis are the main methods currently used to obtain NBP. However, because it is found in very low concentrations in plant tissues, extraction using phenolic and oxygenated solvents is not practical. On the other hand, chemical synthesis results in low bioavailability, solubility, and stability of the compound. For these reasons, bioproduction represents a promising alternative for large-scale NBP production.
AIM 1: The first aim of my final project is to optimize the conditions for the production of the compound NBP.
To achieve this objective, it is necessary to carry out several stages. The general outline of each of these stages is presented below:
STEP 1: Identify the DNA sequence of interest
A literature search was conducted to identify the DNA genetic sequence of the gene involved in the production of the NBP compound. Since NBP is an active compound found in plants of the Apiaceae family, it is produced through a biosynthetic pathway involving several precursor compounds and converting enzymes.
In a study conducted by Chen et al. (2023), a possible biosynthetic pathway for the NBP compound was identified in Ligusticum chuanxiong, in which one of the precursors is a compound called Senkyunolide. This finding was supported by a subsequent study by Nie et al. (2024), using the same plant species, where they determined that both Senkyunolide and Ligustilide are precursors of NBP. Additionally, this study identified the enzyme responsible for converting Senkyunolide into NBP, which they named Lc2OGD2. The DNA genetic sequence of this enzyme was obtained through email correspondence with the respective authors.
STEP 2: Clone the DNA of interest into an Expression Vector
The cloning process of the Lc2OGD2 gene will be carried out using restriction enzymes. The selected expression vector is pET-28a (+) and the host organism is E. coli. Prior to cloning, a codon optimization process will be performed using the bioinformatics tool provided by Twist.